Разнообразие, систематика и филогения амеб семейства Thecamoebidae (Amoebozoa: Discosea) тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Мезенцев Елисей Сергеевич

• Мезенцев Елисей Сергеевич
• кандидат наук
• 2023

Оглавление диссертации кандидат наук Мезенцев Елисей Сергеевич

Актуальность работы

Теоретическая и практическая значимость

Научная новизна

Основные положения, выносимые на защиту:

Апробация и публикации

Личный вклад автора

2. Цель и задачи работы:

3. Обзор литературы

3.1 История изучения семейства Thecamoebidae и его современное положение в системе лобозных амеб

3.2 Состав семейства Thecamoebidae

Род Thecamoeba Fromentel,

T. aesculea Kudryavtsev et Hausmann,

T. quadrilineata (Carter, 1856) Lepçi,

T. similis (Greeff, 1891) Lepçi,

T. sphaeronucleolus (Greeff, 1891) Schaeffer,

T. terricola (Greeff, 1866) Lepçi,

T. striata (Penard, 1890) Schaeffer,

T. munda Schaeffer,

T. hilla Schaeffer,

T. orbis Schaeffer,

T. papyracea (Penard, 1905) Lepçi,

T. rugosa Schaeffer,

T. verrucosa (Ehrenberg, 1838) Schaeffer,

T. (Striamoeba) sparolata Fishbeck et Bovee,

Род Sappinia Dangeard,

Sap. pedata Dangeard,

Sap. diploidea Hartmann and Nägler,

Sap. platani Wylezich, Walochnik, Corsaro, Michel et Kudryavtsev,

Род Stenamoeba Smirnov, Nassonova, Chao et Cavalier-Smith

St. stenopodia (Page, 1969) Smirnov, Nassonova, Chao et Cavalier-Smith

St. amazonica Dykova, Kostka et Peckova,

St. limacina Dykova, Kostka et Peckova,

St. berchidia Geisen, Weinert, Kudryavtsev, Glotova, Bonkowski et Smirnov,

St. sardiniensis Geisen, Weinert, Kudryavtsev, Glotova, Bonkowski et Smirnov,

St. polymorpha Peglar, Nerad et Anderson,

St. dejonckheerei Borquez-Román, Lares-Jiménez, Rodriguez-Anaya, Gonzalez-Galaviz, Ibarra-Gámez,

Casillas-Hernández et Lares-Villa,

Род Stratorugosa Melton et Tekle,

Род Thecochaos Page,

Род Pseudothecamoeba Page,

3.3 Морфология и организация клетки представителей семейства Thecamoebidae

Локомоторная форма

Флотирующая форма

Ядро

Органеллы и цитоплазматические включения

Покровы

Цисты

3.4 Биология текамебид

4. Материал и методики

Изоляция и культивирование

Световая микроскопия

Выделение ДНК, амплификация и филогенетический анализ

5. Результаты и обсуждение

5.1 Видовое разнообразие семейства Thecamoebidae

Thecamoeba aesculea, штамм Та4

Thecamoeba qudrilineata, штамм Та24

Thecamoeba foliovenanda, штамм Та72

Thecamoeba cosmophorea, штамм Та75

Thecamoeba astrologa, штамм Та117

Thecamoeba vumurta, штамм Та

Sappinia diploidea, штамм Sap3

Stenamoeba aeronauta, штамм Stenaero

Vanella primoblina, штамм Та79 (Van79)

5.2 Морфология ядра как таксономический признак амеб рода Thecamoeba

5.3 Виды-двойники и проблема интерпретации «старых» статей

5.4 Реизоляция и изучение рода Thecochaos

5.5 Молекулярная филогения семейства Thecamoebidae

5.6. Морфологические группы видов в пределах семейства Thecamoebidae

5.7. Stenamoeba aeronauta - первый пример скрытого вида в роде Stenamoeba

5.8. Vannella primoblina: стриатный морфотип за пределами семейства Thecamoebida

Основные полученные результаты

Выводы

Благодарности

Список используемой литературы

1. Введение

Введение диссертации (часть автореферата) на тему «Разнообразие, систематика и филогения амеб семейства Thecamoebidae (Amoebozoa: Discosea)»

Актуальность работы

Амёбы семейства Thecamoebidae (текамебиды) широко распространены в различных морских, пресноводных и наземных местообитаниях (Page 1971, 1977). Представителей этой группы можно встретить даже в «экзотичных» местах обитания, таких как горячие источники или гуано летучих мышей (Mulec et al. 2015; Tyml and Dykova 2018; Borquez-Roman et al. 2020; Berrilli et al. 2021), а некоторые виды текамебид могут переходить к амфизойному или даже паразитическому образу жизни (Gelman et al. 2001; Dykova et al. 2010; Borovickova et al. 2019). Характерные очертания клеток текамебид, не образующих дискретных псевдоподий во время локомоции(исключением может до какой-то степени являться род Thecochaos), наличие хорошо заметных складок на поверхности клетки, а также относительная простота изоляции и культивирования делают их удобным объектом для широкого спектра работ: от цитологических исследований до использования в качестве экологических маркеров.

Семейство Thecamoebidae объединяет амеб стриатного, ругозного и языковидного морфотипов, обладающих плотными клеточными покровами, часто описываемыми как «упругие» (Smirnov and Goodkov 1999; Smirnov and Brown 2004). Согласно системе 2011 года, в состав группы входят три рода: центральный род Thecamoeba, а также роды Sappinia и Stenamoeba (Smirnov et al. 2011). В 2019 году, основываясь на молекулярных данных, в состав семейства был включен новый род Stratorugosa (Melton et al. 2019). Совсем недавно нами был исследован, переописан и, на основании морфологических и молекулярных данных, включен в состав семейства Thecamoebidae род Thecochaos, ранее известный только по постоянным препаратам начала XX века (Mesentsev et al. 2022a). К семейству Thecamoebidae по морфологическим данным иногда относят также род Pseudothecamoeba, однако из-за недостатка данных его систематическое положение не ясно и требует дополнительных исследований (Page 1981, 1988; Adl et al. 2019).

На данный момент в состав семейства Thecamoebidae достоверно входят 20 видов амеб, объединенные в пять родов, при этом уровень их изученности очень разный. Для всех видов родов Sappinia, Stenamoeba и единственных подробно описанных видов из родов Stratorugosa и Thecochaos имеются молекулярные данные (Corsaro et al. 2017; Melton et al. 2019; Borquez-Roman et al. 2020; Mesentsev et al. 2022a). В то же время для рода Thecamoeba ощущается сильная нехватка в такого рода данных: из девяти детально описанных видов только для трех имеются сиквенсы гена 18S рРНК. Доступны также сиквенсы генов актина и бета-тубулина некоторых штаммов и транскриптомные данные по виду T. quadrilineata и

неидентифицированному штамму, обозначенному как Thecamoeba sp. Диаметрально противоположная ситуация с родом Stenamoeba. На сегодня описано семь видов стенамеб, для каждого из которых получены молекулярные данные. Однако в базе данных ГенБанк содержится почти в два раза больше последовательностей гена 18s рРНК, обозначенных как «Stenamoeba sp.», или таких, которые по результатам филогенетического анализа относятся к стенамебам. При этом многие из них образуют отдельные ветви на филогенетическом дереве и имеют значительные отличия от последовательностей известных видов. Таким образом, существенная часть разнообразия рода Stenamoeba, по всей видимости, известна лишь по молекулярным данным и не охарактеризована морфологически.

Последние более-менее подробные морфологические исследования большинства видов рода Thecamoeba были проведены более 50 лет назад. Результатом этого исследования стали две работы, которые в совокупности представляют собой ревизию рода Thecamoeba и включают в себя подробные морфологические описания семи видов, хорошо отличимых на светомикроскопическом уровне по очертаниям локомоторной формы и организации внутриядерных структур (Page 1971, 1977). Однако недавние, в том числе и наши, работы показали, что разнообразие текамеб существенно недооценено (Kudryavtsev and Hausmann 2009; Mesentsev and Smirnov 2019, 2021; Mesentsev et al. 2020, 2022б). Скорее всего этот род амеб включает в свой состав несколько десятков видов и в перспективе может быть разделен на несколько самостоятельных таксономических групп. В его состав входят несколько групп видов-двойников, надежно разделить которые можно только с использованием молекулярных данных. Аналогичная ситуация, по всей видимости, наблюдается и в пределах рода Stenamoeba. Более того, ругозный и стриатный морфотипы, как оказалось, могут быть признаком не только амеб семейства Thecamoebida, но и некоторых видов отряда Vannellida. Все эти данные показывают, что семейство Thecamoebidae остается серьёзно недоизученным, и применение современных морфологических и молекулярных методов может существенно обогатить и изменить наши текущие представления о его разнообразии, филогении и систематике.

Теоретическая и практическая значимость

Амебоидные протисты («амебы») являются неотъемлемой частью микробиома как в пресноводных, так и в почвенных местообитаниях, составляя в некоторых типах биотопов до 70% всего разнообразия микроэукариот (Ardnt 1993). Способность амеб к быстрому размножению в благоприятных условиях делает эту группу одной из ключевых в процессе регулирования численности бактерий, грибов и других групп микроскопических организмов в различных биогеоценозах (Anderson 2007; Risse-Buhl et al. 2015; Geisen 2016; Geisen et al. 2018). Амебы способны разрушать бактериальные пленки на поверхности субстратов и играют решающую роль в регуляции роста пленкообразующих бактерий (biofilm-forming bacteria) (Jackson and Jones 1991; Heaton et al. 2001; Martin et al. 2020). Разрушая биопленки, амебы делают составляющих их бактерий доступными и для других групп организмов, что обеспечивает замедление или даже остановку развития биопленок (Jahnke et al. 2007; Anderson 2013). Подобные процессы влекут за собой видоизменение всего сообщества микроорганизмов, и в этом случае амебы выступают в роли ключевого компонента, обеспечивающего формирование сообщества и в значительной степени регулирующего его пространственную структуру и видовой состав.

Представители семейства Thecamoebidae весьма обильны в природных местообитаниях и в основном представлены крупными и среднеразмерными видами (Page 1971, 1977). Их рацион питания весьма широк - от бактерий и грибов и до нитчатых водорослей и других протистов. Это делает их важным компонентом в потоках вещества и энергии в природных экосистемах. В связи с этим изучение видового разнообразия текамебид, оценка их потенциальных местообитаний и «горячих точек» (hotspots) биоразнообразия является важной как с теоретической (разнообразие и объем таксона), так и с практической (роль в природных системах) точек зрения.

Некоторые виды Amoebozoa опасны для человека и домашних животных. Представители ряда родов амеб (Acanthamoeba, Balamuthia, Entamoeba, Vermamoeba, Vannella) и, в частности, рода Sappinia, относящегося к текамебидам, могут вызывать заболевания человека - такие, как кератиты (крайне актуальная проблема в эпоху распространения контактных линз), диарею, амебный менингоэнцефалит (Marciano-Cabral and Cabral 2003; Stanley 2003; Visvesvara et al. 2007; Moran et al. 2022). Необходимо точно идентифицировать патогенные виды амеб (в том числе и текамебид), встреченные в тканях человека и животных, а для этого с очевидностью необходимо прежде всего пополнение баз сиквенсов генов текамебид и изучение проблемы молекулярной дифференциации видов этих амеб. Эта проблема становится особенно

актуальной в свете наших недавних находок видов-двойников в пределах родов Thecamoeba и Stenamoeba (Mesentsev et al. 2022б).

Текамебиды представляют собой одну из ключевых групп в филогенетическом древе Amoebozoa. Доступные сиквенсы их генов SSU представляют собой очень длинные ветви на филогенетическом дереве, а сами сиквенсы зачастую содержат длинные интроны, которые существенно затрудняют амплификацию генов (Mesentsev and Smirnov 2019; Mesentsev et al. 2022а). Подобная особенность делает крайне актуальной задачу пополнения базы сиквенсов генов текамебид и увеличение количества представленных в филогенетическом дереве видов этих амеб.

Наконец, к текамебидам предположительно относят два рода амеб (Pseudothecamoeba и Thecochaos), обладающие совершенно необычными чертами строения клетки по сравнению с типичными представителями семейства Thecamoebidae (Page 1988; Smirnov et al. 2011). В случае, если их принадлежность к этому семейству амеб будет подтверждена, придется пересматривать базовые критерии, ограничивающие семейство, включая возможные морфотипы его представителей, значимость морфологии локомоторной формы, строение и количества ядер в клетке. Это повлияет не только на систематику семейства Thecamoebidae, но будет иметь значение для систематики Amoebozoa в целом.

Научная новизна

В рамках данной работы мы впервые за последние 30 лет года обобщили разрозненные данные по текамебидам, оценили степень изученности их видового разнообразия, описали новые виды текамебид и дополнили информацию о широко распространенных видах. Собрана и исследована коллекция штаммов текамебид, включающая в своей состав более 50 культур, изолированных из различных местообитаний по всему земному шару. В частности, в коллекции представлено не менее 15 морфологических видов рода Thecamoeba, что существенно превышает известное видовое разнообразие этого рода амеб, ряд штаммов из этой коллекции уже описан как новые для науки виды (Mesentsev and Smirnov 2019, 2021; Mesentsev et al. 2020, 2022б). По всем изученным видам были полученные современные светомикроскопические данные, включая фотографии, сделанные с использованием контраста Номарского и качественные видеозаписи движущихся клеток амеб. Для многих видов впервые была детально изучена морфология ядра клетки и прослежена динамика ее изменений по мере развития и старения культуры амеб. Результаты наших исследований показали высокое разнообразие организации ядрышкового материала у видов рода Thecamoeba. Нам удалось обнаружить штамм с исключительно высокой внутривидовой изменчивостью по этому признаку, что ставит новые проблемы в определении видов текамеб на светомикроскопическом уровне (Mesentsev and Smirnov 2021).

Были получены новые молекулярные данные как для уже известных видов, так и для новых для науки видов. Полученные сиквенсы позволили значительно расширить молекулярные базы данных о семействе Thecamoebidae, и частности - его центрального рода -Thecamoeba, в котором недостаток молекулярных данных ощущается наиболее сильно. Полученные молекулярные данные позволили оценить генетическое разнообразие текамебид. Анализ молекулярных данных позволил обнаружить скрытое разнообразие среди широко распространенных морфологических видов текамебид. Нам удалось изолировать и изучить представителя одного из двух загадочных родов амеб, ранее лишь предположительно отнесенных к семейству Thecamoebidae. Мы обнаружили редкий и малоизвестный вид амеб -Thecochaos fibrillosum, который не попадался исследователям более 100 лет и был изолирован нами из проб почвы черневой тайги южной Сибири. Молекулярная филогения показала, что этот род амеб достоверно относится к семейству Thecamoebidae. В рамках этой работы мы использовали практически весь арсенал методов молекулярных исследований одиночных клеток и наглядно показали, что на современном уровне развития технологий даже одиночных клеток амебы достаточно для того, чтобы получить полный набор данных об изучаемом

организме, включая светомикроскопические, ультраструктурные, и молекулярные, в том числе - геномные данные.

В рамках нашей работы в пределах морфологических видов текамебид впервые были обнаружены примеры скрытого разнообразия на генетическом уровне и предложена концепция морфологических групп видов текамебид, с учетом наличия видов-двойников (МеБе^еу й а1. 2022б). Использование в качестве наименований названий групп видов позволит избежать путаницы при рутинном определении текамебид светомикроскопическими методами.

Выполненное исследование позволило вывести на современный уровень изучение амеб семейства ТЬесашоеЫёае, продемонстрировало высокий уровень видового разнообразия этой группы амеб и четко показало, что корректная идентификация видов в подавляющем большинстве случаев возможна только с использованием молекулярных данных. Этот факт необходимо учитывать при интерпретации результатов работ, в которых виды текамебид определяли только на светомикроскопическом уровне, поскольку авторы могли иметь дело с видами-двойниками, плохо отличимыми (или вообще неотличимыми) по морфологическим признакам.

Основные положения, выносимые на защиту:

1. Разнообразие наземных и пресноводных представителей семейства Thecamoebidae остается сильно недооцененным, по всей видимости, эта группа содержит ещё много неизвестных видов амеб.

2. Наземные местообитания, в частности - поверхность высохших травянистых растений, кора деревьев и иные растительные субстраты представляют собой «очаги разнообразия» текамебид, и в особенности - представителей рода Thecamoeba.

3. Традиционно используемые для идентификации голых амеб морфологические признаки (размер, локомоторная форма, строение ядра) не всегда являются надежными и достаточными признаками для дифференциации видов текамебид.

4. Многие широко известные виды текамебид, описанные на морфологическом уровне, могут представлять собой комплексы видов-двойников (sibling species). Надежно отличить их друг от друга можно только с использованием молекулярных данных, в частности - сиквенса гена 18S рРНК.

Апробация и публикации

По материалам диссертации опубликовано 7 научных статей в журналах, индексируемых системами WoS и/или Scopus, и 6 публикаций в материалах международных и всероссийских конференций.

Основные положения и научные итоги диссертации были изложены в докладах на научных конференциях:

- VII European Congress of Protistology (ECOP, 5-10.9.2015)

- Moscow forum "Protist 2016" (6-10.6.2016)

- XXII meeting of the International Society of Evolutionary Protistology (ISEP, 27.51.6.2018)

- Клеточная биология: проблемы и перспективы (2-6.10.2017)

- VII молодёжная школа-конференция по молекулярной и клеточной биологии (1215.10.2020)

Публикации по материалам диссертации:

Mesentsev, Y., Bondarenko, N., Kamyshatskaya, O., Nassonova. E., Glotova, A., Loiko, S., Istigechev, G., Kulemzina, A., Abakumov, E., Rayko, M., Lapidus, A. and Smirnov, A., 2022. Thecochaos is not a myth: study of the genus Thecochaos (Amoebozoa, Discosea) - a rediscovered group of lobose amoeba, with short SSU gene. Org. Divers. Evol. https://doi.org/10.1007/s13127-022-00581-9

Mesentsev, Y., Kamyshatskaya, O., Nassonova, E., Smirnov, A., 2022. Thecamoeba vumurta n. sp. (Amoebozoa, Discosea, Thecamoebida) from freshwater pond sediment - a sibling species of T. striata (Penard, 1890) Schaeffer, 1926. Eur. J. Protistol. 83, 125866. https://doi.org/10.1016/j.ejop.2022.125866

Mesentsev, Y., Smirnov, A., 2021. Thecamoeba astrologa n. sp. - a new species of the genus Thecamoeba (Amoebozoa, Discosea, Thecamoebida) with an unusually polymorphic nuclear structure. Eur. J. Protistol. 81, 125837. https://doi.org/10.1016/j.ejop.2021.125837

Mesentsev, Y., Bondarenko, N., Nassonova, E., Smirnov, A., 2021. Vannellaprimoblina n. sp. - an unusual species of the genus Vannella (Amoebozoa, Discosea, Vannellida) with pronounced dorsal ridges and folds. Eur. J. Protistol. 77, 125757. https://doi.org/10.1016/j.ejop.2020.125757

Mesentsev, Y., Kamyshatskaya, O., Smirnov, A., 2020. Thecamoeba foliovenanda n. sp. (Amoebozoa, Discosea, Thecamoebida) - One more case of sibling species among amoebae of the genus Thecamoeba. Eur. J. Protistol. 76, 125716. https://doi.org/10.1016/j.ejop.2020.125716

Mesentsev, Y.S., Smirnov, A.V., 2019. Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) - An example of sibling species within the genus Thecamoeba. Eur. J. Protistol. 67, 132-141. https://doi.org/10.1016/j.ejop.2018.12.003

Kamyshatskaya, O., Mesentsev, Y., Smirnov, A., Michel, R., Walochnik, J., Nassonova, E., 2018. Fine structure of Thecamoeba quadrilineata strain CCAP 1583/10 (Amoebozoa, Discosea, Thecamoebida), the host of Nucleophaga amoebae (Opisthosporidia). Protistology 12, 191-201. https://doi.org/10.21685/1680-0826-2018-12-4-4

Личный вклад автора

Сбор проб из разных географических точек независимо производился автором в Ленинградской области, Санкт-Петербурге, Котке (Финляндия) и ХМАО. Отдельные пробы были доставлены Хафизовой Галиной из Белгородской области; Камышацкой Оксаной из Ижевска; Вотиновой Валентиной из Кисловодска; Сергеем Лойко, Алексеем Абакумовым и Георгием Истигечевым - из Томской области.

Основная экспериментальная часть работы была выполнена автором, а именно - высев проб, установление и ведение лабораторных культур амеб, световая микроскопия клеток и постобработка полученных изображений, сбор и подготовка клеток для выделения тотальной ДНК из культур и одиночных клеток, выделение тотальной ДНК, ПЦР, качественная оценка и очистка ПЦР-продуктов, обработка и анализ полученных сиквенсов, филогенетический анализ на основе полученных последовательностей генов.

Исключение составили: полногеномная амплификация образцов тотальной ДНК изолятов Van79 и Tch1 были выполнены Насоновой Еленой Станиславовной. Использованный в анализе сиквенс гена 18s рРНК штамма Thecamoeba striata CCAP 1583/4, был получены Насоновой Еленой Станиславовной и Жозе Фарни в 2001 году, но был принят ими за сиквенс контаминанта. Корректно опознан и аттрибутирован он был автором. Подготовка библиотек и выполнение NGS-секвенирования, а также секвенирование по Сенгеру выполняли в ресурсном центре Научного парка СПбГУ «Развитие молекулярных и клеточных технологий». Очистка и сборка данных NGS-секвенирования (изоляты Van 79 и Tch1) осуществлялась Натальей Бондаренко и Михаилом Райко, им же было подготовлено мультигенное выравнивание (изолят Tch1).

2. Цель и задачи работы:

Цель работы: изучить разнообразие, систематику и филогению амёб семейства ТЬесатоеЫёае и подготовить материалы для проведения ревизии и монографического описания этого семейства амеб.

Задачи работы:

1. Изолировать амеб, относящихся к семейству ТЬесатоеЫёае из природных местообитаний, по возможности установить их в лабораторных культурах.

2. Изучить изолированные штаммы методами световой микроскопии, идентифицировать их или подготовить материалы для последующего их описания как новых для науки видов.

3. Амплифицировать и секвенировать ген 18б рРНК выделенных штаммов и провести филогенетический анализ.

4. Сопоставить данные филогенетического анализа с данными по морфологии изолированных штаммов. Оценить значимость и надежность традиционно используемых для идентификации амеб семейства ТЬесатоеЫёае морфологических признаков.

3. Обзор литературы

3.1 История изучения семейства Thecamoebidae и его современное положение в системе

лобозных амеб

До начала XX века большинство работ посвященных систематике амебоидных организмов представляли общие системы «корненожек», в рамках которых исследователей больше интересовало взаиморасположение групп различных раковинных амеб, фораминифер и «лучевиков» (Leidy 1879; Butcschli 1882; Penard 19G2; Аверинцев 19G6). Лишь в начале XX века в системе начинают появляться отдельные более-менее четко очерченные семейства голых лобозных амеб (Poche 1913; Schaeffer 1926).

В ранних работах можно встретить таксоны под именем «Thecamoebida» и «Thecamoebina». Эти группы содержат раковинных амеб и, соответственно, противопоставляются группам «Gymnamoebida» и «Gymnamoebina», в состав которых входят в основном голые лобозные амебы (Аверинцев 19G6). Однако при дальнейшем развитии системы амебоидных организмов эти названия перестали использоваться, и они не являются прямыми предшественниками современного названия семейства Thecamoebidae.

На основании комплексного анализа известных и новых видов амеб Шаффером была разработана новая система голых лобозных амеб (Schaeffer 1926). Наибольшее внимание при построении системы было уделено форме тела амебы, которую она принимает во время движения. Эта идея «локомоторной формы» - формы амебы в движении, как одного из важнейших признаков организации клетки дошла до наших дней и, в частности, была использована в работах посвященных морфотипам лобозных амеб (Smirnov and Goodkov 1999, Smirnov and Brown 2GG4). Именно в работе 1926 года Шаффер установил семейство Thecamoebidae. Новый таксон был расположен в рамках подотряда Amœbœa Ehrenberg, 183G, отряда Lobosa Carpenter, 1861, класса Rhizopoda Siebold, 1845. В состав семейства Thecamoebidae Шаффер включил род Thecamoeba Fromentel, 1874 и установленный им новый род Rugipes. Диагноз семейства Thecamoebidae sensu Schaeffer 1926 звучал следующим образом: «Эти амебы не образуют псевдоподий во время локомоции. В общих чертах, локомоторная форма овальная до круглой, с небольшими вариациями. Продольные гребни встречаются почти во всех видах» (Schaeffer 1926, стр. 83).

Шаффер указывал, что «при нарушении», то есть при флотировании (по Page 1977), амебы рода Rugipes образуют длинные псевдоподии и принимают «истинно лучевую форму», что и является главным отличием их от представителей рода Thecamoeba. Однако, при

движении по субстрату особых отличий в локомоторной форме представители этих родов не имеют. При этом Шаффер считал овальную форму тела и толстую эктоплазму, образующую продольные гребни, основными эволюционными трендами текамебид. Поскольку представители рода Rugipes способны формировать дискретные псевдоподии, хотя только во время флотирования, Шаффер считал род Rugipes предковым по отношению к Thecamoeba, представители которого никогда не образуют псевдоподий (Рис. 1).

Новый род Rugipes включал в свой состав два вида: R. vivax и R. bilzi. Впоследствии этот род был расформирован, а его представители были перемещены в другие рода: R. vivax сначала включен Бови в 1965 году в новый род Vannella входящий в состав подсемейства Vannellina семейства Flabellulidae, затем Пэйджем в 1969 году в новый род Platyamoeba также вошедший в состав подсемейства Vannellina (Bovee 1965; Page 1969). Дальнейшие изменения были внесены Сойером в 1975 году, когда внутри семейства Thecamoebidae был создан новый род Clydonella; и R. vivax было дано новое название Clydonella vivax (Sawyer 1975). Второй вид - R. bilzi в 1971 году был перенесен в состав рода Thecamoeba (Page 1971), однако представители этого вида ни разу не были достоверно ре-изолированы из природных местообитаний, в связи с чем его таксономическое положение остается неясным и в целом валидность этого вида остается под вопросом.

В последующем в первой половине XX века было опубликовано большое количество работ, предлагающих разнообразные системы амеб. Все они базировались на различных способах анализа морфологических признаков амебоидных организмов, хотя в целом ориентировались на морфологию клеток (Kudo 1939; Raabe 1948; Hall 1953; Reichenov 1953). Попытку создать принципиально новую систему предпринял в 1952 году Сингх (Singh 1952) и далее развил в 1953 году Шаттон. Сингх обращает особое внимание на особенности митоза амеб, и основываясь на этом признаке выделяет новые семейства. Далее Шаттон, считая наличие жгутиков самым базальным признаком для построения системы амеб, установил два новых подотряда: Mastigogenina («со жгутиковой стадией») и Amastigogenina («без жгутиковой стадии»). В этой системе семейство Thecamoebidae входило в состав подотряда Amastigogenina, при этом его родовой состав не изменился (Chatton 1953). Однако дальнейшее развитие системы, основанной на особенностях митоза, стерло практически все ранее установленные ранее семейства амеб. В работе 1970-го Сингх и Дас предложили новую систему разделив отряд Amoebida всего на три семейства (Singh and Das 1970). При этом в обновленной системе не были упомянуты многие рода голых амеб, отчего проследить судьбу многих представителей семейства Thecamoebidae не представляется возможным.

Рисунок 1. Схемы и рисунки из работы Шаффера (из Schaeffer 1926). А - Схема филогенетических отношений крупных амёб. Б - Схема последовательного изменения морфологии движущейся клетки Thecamoeba rugosa (A), флотирующая форма (B) и «необычная» форма клетки в старой культуре (C). В - Схема последовательного изменения морфологии движущейся клетки Rugipes vivax (A), различные варианты заднего конца (B-D), выросты на переднем конце (E), очертания клетки и направление тока цитоплазмы при смене движения клетки (F), и схема перехода клетки из флотирующей стадии в прикрепленную (G). Г - Локомоторная форма Rugipes vivax. Д - Локомоторная форма Thecamoeba hilla. Е -Локомоторная форма Thecamoeba orbis. З - Локомоторная форма Thecamoeba munda.

В начале второй половины XX века в серии работ Бови и Яна система амеб вновь претерпела существенные изменения (Bovee 1954, 1970, 1972; Bovee and Jahn 1960, 1966; Jahn and Bovee 1965; Jahn et al. 1974). В системе Бови и Яна 1966 года голые лобозные амебы в рамках надотряда Lobida были разделены на три отряда. Основным признаком при выделении этих отрядов был механизм амебоидного движения и особенности тока цитоплазмы в псевдоподиях клеток. Семейство Thecamoebidae было единственным входившим в состав подотряда Rugina нового отряда Thecida. Помимо изменения положения в системе, также изменился и состав семейства, в котором остался только род Thecamoeba, а род Rugipes был переведен в состав сестринского семейства Striamoebidae. Диагноз семейства Thecamoebidae совпадал с диагнозом подотряда и выглядел следующим образом: «Крупные амебы, образующие большое количество складок во время движения. Складки расположены на дорсальной стороне и расположены более-менее параллельно. Псевдоподии представляют собой только тупые выросты» (Bovee and Jahn 1966, стр. 233; Jahn et al. 1974, стр. 430).

Состав рода Thecamoeba также был сильно изменен: многие виды, ранее входившие в его состав, были включены в состав нового рода Striamoeba семейства Striamoebidae подотряда Striatina. Подотряд Striatina так же как и подотряд Rugina входил в состав отряда Thecida. Диагноз семейства Striamoebidae, идентичный диагнозу подотряда Striatina звучал так: «Маленькие амебы с отчетливыми параллельными дорсальными продольными гребнями при локомоции; псевдоподии формируются редко, но только как тупые выросты». Подобное выделение групп амеб «крупных с морщинами» и «маленьких с гребнями» также нашло свое в современной классификации локомоторных форм и соответствует ругозному и стриатному морфотипам, за исключением того, что в классификации морфотипов не учитываются размерные характеристики. Авторы в работе не указали четкой границы между «крупными» и «мелкими» амебам; граница в 100 мкм появилась в более поздней работе, которая представляет собой определитель амеб, составленный Бови в 1985 году (Bovee 1985). Таким образом, при всех новых и сохранившихся старых таксонах в этих работах представители семейства Thecamoebidae были распределены по разным группам в рамках отряда Thecida (Рис. 2).

Рисунок 2. Схематичное изображение текамебид (из Bovee and Sawyer 1979). A - Флотирующая и локомоторные формы «Thecamoeba terricola (verrucosa)». Б -Флотирующая и локомоторные формы «Striamoeba hilla». В - Флотирующая и локомоторные формы «Striamoeba rugosa». Г - Флотирующая и локомоторные формы «Striamoeba orbis». Д -Локомоторные формы «Striamoeba munda».

Список литературы диссертационного исследования кандидат наук Мезенцев Елисей Сергеевич, 2023 год

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Saint Petersburg State University

As Manuscript

Mesentsev Yelisei Sergeevich

DIVERSITY, TAXONOMY AND PHYLOGENY OF AMOEBAE OF THE FAMILY THECAMOEBIDAE (AMOEBOZOA: DISCOSEA)

Scientific specialization: 1.5.12. Zoology Thesis for the degree of Candidate of Biological Sciences Translation from Russian

Supervisor: docent, Ph.D. Smirnov A. V.

Saint Petersburg 2022

Contents

1. Introduction........................................................................................................................150

Relevance of the work......................................................................................................................150

The theoretical and practical significance of the work.................................................................152

The scientific novelty of work.........................................................................................................154

Principal findings to be considered.................................................................................................156

Publications and approbation of work...........................................................................................157

Author's personal contribution.......................................................................................................158

2. Goal and tasks:...................................................................................................................159

3. Literature review................................................................................................................160

3.1 History of the study of the family Thecamoebidae and its current position in the lobose amoebae system.............................................................................................................................................160

3.2 Genera of the family Thecamoebidae.......................................................................................171

Genus Thecamoeba Fromentel, 1874............................................................................................ 171

T. aesculea Kudryavtsev et Hausmann, 2009........................................................................................... 175

T. quadrilineata (Carter, 1856) Lepçi, 1960............................................................................................. 175

T. similis (Greeff, 1891) Lepçi, 1960........................................................................................................ 176

T. sphaeronucleolus (Greeff, 1891) Schaeffer, 1926................................................................................ 177

T. terricola (Greeff, 1866) Lepçi, 1960.................................................................................................... 179

T. striata (Penard, 1890) Schaeffer, 1926................................................................................................. 180

T. munda Schaeffer, 1926......................................................................................................................... 181

T. hilla Schaeffer, 1926............................................................................................................................. 181

T. orbis Schaeffer, 1926............................................................................................................................ 182

T. papyracea (Penard, 1905) Lepçi, 1960................................................................................................. 182

T. rugosa Schaeffer, 1926......................................................................................................................... 182

T. verrucosa (Ehrenberg, 1838) Schaeffer, 1926...................................................................................... 183

T. (Striamoeba) sparolata Fishbeck et Bovee, 1993 ................................................................................ 183

Genus Sappinia Dangeard, 1896.................................................................................................... 184

Sap. pedata Dangeard, 1896..................................................................................................................... 184

Sap. diploidea Hartmann and Nägler, 1908.............................................................................................. 185

Sap. platani Wylezich, Walochnik, Corsaro, Michel et Kudryavtsev, 2015............................................187

Genus Stenamoeba Smirnov, Nassonova, Chao et Cavalier-Smith 2007...................................... 188

St. stenopodia (Page, 1969) Smirnov, Nassonova, Chao et Cavalier-Smith 2007................................... 189

St. amazonica Dykova, Kostka et Peckova, 2010.....................................................................................189

St. limacina Dykova, Kostka et Peckova, 2010........................................................................................190

St. berchidia Geisen, Weinert, Kudryavtsev, Glotova, Bonkowski et Smirnov, 2014.............................190

St. sardiniensis Geisen, Weinert, Kudryavtsev, Glotova, Bonkowski et Smirnov, 2014.........................190

St. polymorpha Peglar, Nerad et Anderson, 2016.....................................................................................191

St. dejonckheerei Borquez-Román, Lares-Jiménez, Rodriguez-Anaya, Gonzalez-Galaviz, Ibarra-Gámez,

Casillas-Hernández et Lares-Villa, 2020................................................................................................................ 191

Genus Stratorugosa Melton et Tekle, 2019...................................................................................192

Genus Thecochaos Page, 1981......................................................................................................193

Genus Pseudothecamoeba Page, 1988 .......................................................................................... 194

3.3 Cell morphology and organisation of amoebae of the family Thecamoebidae.....................195

Locomotive form...........................................................................................................................195

Floating form.................................................................................................................................19б

Nucleus..........................................................................................................................................197

Organelles and cytoplasmic inclusions..........................................................................................200

Cell surface....................................................................................................................................201

Cysts...............................................................................................................................................202

3.4 Biology of thecamoebids ............................................................................................................ 203

4. Material and methods ........................................................................................................ 205

Isolation and cultivation .................................................................................................................. 205

Light microscopy..............................................................................................................................206

DNA extraction, amplification and phylogenetic analysis............................................................ 207

5. Results and discussion........................................................................................................209

5.1 Species diversity in the family Thecamoebidae.......................................................................209

Thecamoeba aesculea, strain Ta4..................................................................................................218

Thecamoeba quadrilineata, strain Ta24........................................................................................221

Thecamoeba foliovenanda, strain Ta72.........................................................................................223

Thecamoeba cosmophorea, strain Ta75 ........................................................................................22б

Thecamoeba astrologa, strain Ta117.............................................................................................228

Thecamoeba vumurta, strain Ta130...............................................................................................235

Sappinia diploidea, strain Sap3.....................................................................................................238

Stenamoeba aeronauta, strain Stenaero.........................................................................................240

Vannella primoblina, strain Ta79 (Van79)....................................................................................243

5.3 Sibling species and the problem of interpreting "old" articles .............................................. 248

5.4 Re-isolation and study of the genus Thecochaos.....................................................................251

5.5 Molecular phylogeny of the family Thecamoebidae ............................................................... 257

5.6 Morphological groups of species within the family Thecamoebidae.....................................261

5.7. Stenamoeba aeronauta - the first example of a hidden species in the genus Stenamoeba... 262

5.8. Vannella primoblina: rugose morphotype outside the Thecamoebida family.....................263

Main results obtained ............................................................................................................. 265

Conclusions

266

Acknowledgements ................................................................................................................. 267

References ............................................................................................................................... 268

150

1. Introduction

Relevance of the work

Amoebae of the family Thecamoebidae (thecamoebids) are widely distributed in various marine, freshwater and terrestrial habitats (Page 1971, 1977). They can even be found in "exotic" habitats such as hot springs or bat guano (Mulec et al. 2015; Tyml and Dykova 2018; Borquez-Roman et al. 2020; Berrilli et al. 2021), and some species may switch to amphizoic or parasitic lifestyles (Gelman et al. 2001; Dykova et al. 2010; Borovickova et al. 2019). The characteristic outline of thecamoebids that not form discrete pseudopodia during active directed locomotion (the genera Thecochaos and Pseudothecamoeba may be an exception to some extent), the presence of clearly visible folds on the cell surface, and the relative ease of isolation and culturing make them a convenient object for a wide range of work: from cytological studies to use as ecological markers.

The family Thecamoebidae comprises amoebae belonging to the striate, rugose and lingulate morphotypes with dense cell coat often described as "elastic" (Smirnov and Goodkov 1999; Smirnov and Brown 2004). According to the 2011 classification, the family included three genera: the central genus Thecamoeba, and the genera Sappinia and Stenamoeba (Smirnov et al. 2011). In 2019, a new genus Stratorugosa was added to the family based on molecular data (Melton et al. 2019). More recently, the genus Thecochaos, previously known only from permanent preparations of the early 20th century, was investigated, redescribed and added to the family Thecamoebidae based on morphological and molecular data (Mesentsev et al. 2022a). The genus Pseudothecamoeba is also sometimes included in the family Thecamoebidae on the basis of morphological data, but its systematic position is not clear due to lack of data and requires further research (Page 1981, 1988; Adl et al. 2019).

Currently, the family Thecamoebidae reliably comprises 20 species of amoebae, grouped into five genera, with very different completeness of data on them. Molecular data are available for all species in the genera Sappinia, Stenamoeba, and one species from the genera Stratorugosa and Thecochaos (Corsaro et al. 2017; Melton et al. 2019; Borquez-Roman et al. 2020; Mesentsev et al. 2022a). At the same time, for the genus Thecamoeba, there is a severe lack of such data: there are 18s rRNA gene sequences only for three out of nine species described in detail. Also, there are sequences of actin and beta-tubulin genes from some strains and transcriptome data for T. quadrilineata and for an unidentified strain named as Thecamoeba sp. The situation with the genus Stenamoeba is diametrically opposed. Seven Stenamoeba species have been described, and molecular data have been obtained for each. However, the GeneBank database contains almost twice as many 18s rRNA gene sequences named as "Stenamoeba sp.", or those that are phylogenetically related to Stenamoeba.

Moreover, many of them form separate branches on the phylogenetic tree and have significant differences from the sequences of known species. Thus, a significant part of the diversity of the genus Stenamoeba appears to be known only from molecular data and is not characterized morphologically.

The last detailed morphological studies of most species of the genus Thecamoeba were made out more than 50 years ago (Page 1971, 1977). This study resulted in two articles that together are a revision of the genus Thecamoeba and comprise detailed morphological descriptions of seven species, well distinguishable at the light-microscopic level by the locomotive form and organization of intranuclear structures. However, recent work, including ours, has shown that the diversity of Thecamoeba is significantly underestimated (Kudryavtsev and Hausmann 2009; Mesentsev and Smirnov 2019, 2021; Mesentsev et al. 2020, 2022b). Most likely, this genus of amoebae comprises several dozen species and may be subdivided into several distinct taxonomic groups in the future. The genus Thecamoeba includes several groups of sibling species, which can only be reliably identified using molecular data. A similar situation seems to exist within the genus Stenamoeba. Moreover, the rugose and striate morphotypes appeared to be a feature not only of amoebae of the family Thecamoebidae, but also of some species of the order Vannellida. All these data show that the family Thecamoebidae remains seriously understudied and the application of modern morphological and molecular methods can significantly enrich and change our current understanding of its diversity, phylogeny and systematics.

The theoretical and practical significance of the work

Amoeboid protists ("amoebae") are an integral part of the microbiome in both freshwater and soil habitats, making up to 70% of the total diversity of microeukaryotes in some habitat types (Ardnt 1993). The ability of amoebae to reproduce rapidly under favourable conditions makes this group one of the key ones in regulating the number of bacteria, fungi and other groups of microscopic organisms in various biogeocenoses (Anderson 2008; Risse-Buhl et al. 2015; Geisen et al. 2016, 2018). Amoebae are capable of destroying bacterial films on the surface of substrates and play a crucial role in regulating the growth of biofilm-forming bacteria (Jackson and Jones 1991; Heaton et al. 2001; Martin et al. 2020). By destroying biofilms, amoebae make their constituent bacteria available to other groups of organisms, thus slowing or even stopping biofilm development (Jahnke et al. 2007; Anderson 2013). Such processes lead to the modification of the entire microbial community, in which case the amoebae are a key component in community formation and largely regulate its spatial structure and species composition.

Amoebae of the family Thecamoebidae are very abundant in natural habitats and mainly represented by large and medium-sized species (Page 1971, 1977). Their diet is very broad, ranging from bacteria and fungi to filamentous algae and other protists. This makes them an important component of the matter and energy flows in natural ecosystems. In this context, studying the species diversity of the Thecamoebidae, assessing their potential habitats and biodiversity hotspots is important from both a theoretical (diversity and size of the taxon) and a practical (role in natural systems) points of view.

Some Amoebozoa species are dangerous for humans and domestic animals. Species of several genera of amoebae (Acanthamoeba, Balamuthia, Entamoeba, Vermamoeba, Vannella) and, in particular, the genus Sappinia, related to thecamoebids, can cause human diseases such as keratitis (a highly relevant problem in the era of contact lenses), diarrhoea, amoebic meningoencephalitis (Marciano-Cabral and Cabral 2003; Stanley 2003; Visvesvara et al. 2007; Moran et al. 2022). The pathogenic amoeba species (including thecamoebids) found in human and animal tissues need to be accurately identified. This clearly requires first the replenishment of the thecamoebid gene sequences databases and the study of the problem of molecular differentiation of these amoeba species. This problem becomes particularly relevant in the light of our recent findings of sibling species within the genera Thecamoeba and Stenamoeba (Mesentsev et al. 2022b).

Thecamoebidae represent one of the key groups in the Amoebozoa phylogenetic tree. The available 18s rRNA gene sequences of their genes form very long branches on the phylogenetic tree. Moreover, the sequences themselves often contain long introns that make gene amplification very difficult (Mesentsev and Smirnov 2019; Mesentsev et al. 2022a). This feature makes the task of

updating the sequences base and increasing the number of thecamoebids represented in the phylogenetic tree extremely urgent.

Finally, two genera of amoebae (Pseudothecamoeba and Thecochaos) with quite unusual cell structure features compared with the typical species of thecamoebids are presumed to belong to the Thecamoebidae (Page 1988; Smirnov et al. 2011). If their belonging to this family of amoebae is confirmed, the basic criteria restricting the family would have to be reconsidered, including possible morphotypes of its species, the importance of locomotive morphology, structure and number of nuclei in the cell. This will affect not only the systematics of the family Thecamoebidae, but will have implications for Amoebozoa systematics as a whole.

The scientific novelty of work

In this work, for the first time in the last 30 years, we have summarised scattered data on the Thecamoebidae, evaluated the degree of study of their species diversity, described new species and supplemented information on widespread species. A collection of thecamoebid strains, including more than 50 cultures isolated from different habitats around the globe, has been collected and studied. In particular, the collection includes at least 15 morphological species of the genus Thecamoeba, which significantly exceeds the known species diversity of this genus, and some strains from this collection have already been described as new species (Mesentsev and Smirnov 2019, 2021; Mesentsev et al. 2020, 2022b). For all species studied, modern light-microscopic data were obtained, including photographs taken using Nomarski contrast and high-quality video recordings of moving amoeba cells. For many species, for the first time, the morphology of the cell nucleus was studied in detail and the dynamics of its changes with development and ageing of the amoeba culture were traced. The results of our studies showed a high diversity of the organization of the nucleus material in species of the genus Thecamoeba. We were able to find a strain with extremely high intraspecific variability of this sign, which poses new problems in the identification of Thecamoeba species at the light microscopic level (Mesentsev and Smirnov 2021).

New molecular data were obtained for both already known species and for new species. The sequences obtained have significantly expanded the molecular databases on the family Thecamoebidae and in particular its central genus, Thecamoeba, where the lack of molecular data is most severe. The molecular data obtained made it possible to estimate the genetic diversity of Thecamoebidae. The analysis of molecular data revealed a cryptic diversity among the widespread morphological species of Thecamoeba. We were able to isolate and study a species of one of two enigmatic genera of amoebae, previously only supposedly assigned to the family Thecamoebidae. We found a rare and little-known species of amoeba, Thecochaos fibrillosum, which has not come across to researchers for more than 100 years and was isolated by us from soil samples of chernevaya taiga of southern Siberia. Molecular phylogeny showed that this genus of amoeba reliably belongs to the family Thecamoebidae. Within this work we used almost the entire arsenal of methods for molecular studies of single cells and clearly showed that at the modern level of technology even a single amoeba cell is enough to obtain a full set of data on the studied organism, including light microscopic, ultrastructure, and molecular, including genomic data.

Within our work, examples of cryptic diversity were discovered for the first time within the morphological species of Thecamoebidae and the concept of morphological groups of Thecamoebidae species was proposed, given the presence of sibling species (Mesentsev et al 2022b). The use of

species group names will avoid confusion in routine identification of thecamoebids by light-microscopic methods.

This study brought the study of amoebae of the family Thecamoebidae to the modern level, demonstrated a high level of species diversity of this group of amoebae, and clearly demonstrated that correct identification of species in the vast majority of cases is possible only with the use of molecular data. This fact should be considered when interpreting the results of works in which the species of Thecamoebidae were identified only on the light microscopic level, because the authors could be dealing with sibling species, poorly distinguishable (or not distinguishable at all) by morphological features.

Principal findings to be considered

1. The diversity of terrestrial and freshwater amoebae of the family Thecamoebidae remains greatly underestimated; apparently, this group contains many more unknown species of amoebae.

2. Terrestrial habitats, in particular the surface of dried herbaceous plants, tree bark and other plant substrates, are "hotspots of diversity" for thecamoebids, and in particular for amoebae of the genus Thecamoeba.

3. The morphological characters traditionally used for identification of naked amoebae (size, locomotive form, nucleus structure) are not always reliable and sufficient characters for differentiating thecamoebid species.

4. Many well-known thecamoebid species described at the morphological level can represent complexes of sibling species. They can be reliably distinguished from each other only using molecular data, in particular, the 18S rRNA gene sequence.

Publications and approbation of work

On the materials of the thesis 7 scientific articles were published in the journals indexed by WoS and/or Scopus, and 6 publications in the proceedings of international and Russian conferences.

The main points and scientific findings of the thesis have been presented in conference reports:

- VII European Congress of Protistology (ECOP, 5-10.9.2015)

- Moscow forum "Protist 2016" (6-10.6.2016)

- XXII meeting of the International Society of Evolutionary Protistology (ISEP, 27.51.6.2018)

- Cell biology: problems and prospects (2-6.10.2017)

- VII youth school-conference on molecular and cellular biology (12-15.10.2020)

Publications based on dissertation materials:

Mesentsev, Y., Bondarenko, N., Kamyshatskaya, O., Nassonova. E., Glotova, A., Loiko, S., Istigechev, G., Kulemzina, A., Abakumov, E., Rayko, M., Lapidus, A. and Smirnov, A., 2022. Thecochaos is not a myth: study of the genus Thecochaos (Amoebozoa, Discosea) - a rediscovered group of lobose amoeba, with short SSU gene. Org. Divers. Evol. https://doi.org/10.1007/s13127-022-00581-9

Mesentsev, Y., Kamyshatskaya, O., Nassonova, E., Smirnov, A., 2022. Thecamoeba vumurta n. sp. (Amoebozoa, Discosea, Thecamoebida) from freshwater pond sediment - a sibling species of T. striata (Penard, 1890) Schaeffer, 1926. Eur. J. Protistol. 83, 125866. https://doi.org/10.1016/j.ejop.2022.125866

Mesentsev, Y., Smirnov, A., 2021. Thecamoeba astrologa n. sp. - a new species of the genus Thecamoeba (Amoebozoa, Discosea, Thecamoebida) with an unusually polymorphic nuclear structure. Eur. J. Protistol. 81, 125837. https://doi.org/10.1016/j.ejop.2021.125837

Mesentsev, Y., Bondarenko, N., Nassonova, E., Smirnov, A., 2021. Vannella primoblina n. sp. - an unusual species of the genus Vannella (Amoebozoa, Discosea, Vannellida) with pronounced dorsal ridges and folds. Eur. J. Protistol. 77, 125757. https://doi.org/10.1016/j.ejop.2020.125757

Mesentsev, Y., Kamyshatskaya, O., Smirnov, A., 2020. Thecamoeba foliovenanda n. sp. (Amoebozoa, Discosea, Thecamoebida) - One more case of sibling species among amoebae of the genus Thecamoeba. Eur. J. Protistol. 76, 125716. https://doi.org/10.1016/j.ejop.2020.125716

Mesentsev, Y.S., Smirnov, A.V., 2019. Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) - An example of sibling species within the genus Thecamoeba. Eur. J. Protistol. 67, 132-141. https://doi.org/10.1016/j.ejop.2018.12.003

Kamyshatskaya, O., Mesentsev, Y., Smirnov, A., Michel, R., Walochnik, J., Nassonova, E., 2018. Fine structure of Thecamoeba quadrilineata strain CCAP 1583/10 (Amoebozoa, Discosea, Thecamoebida), the host of Nucleophaga amoebae (Opisthosporidia). Protistology 12, 191-201. https://doi.org/10.21685/1680-0826-2018-12-4-4

Author's personal contribution

Samples from different geographical locations were independently collected by the author in the Leningrad Region, St. Petersburg, Kotka (Finland) and the Khanty-Mansiysk Autonomous Region. Separate samples were collected and delivered by Galina Khafizova from Belgorod oblast; Oksana Kamyshatskaya from Izhevsk; Valentina Votinova from Kislovodsk; Sergey Loiko, Alexey Abakumov and Georgiy Istigechev from Tomsk oblast.

The main experimental part of the work was carried by the author, namely, sample seeding, establishment and maintenance of laboratory cultures of amoebae, light microscopy of cells and postprocessing of the obtained images, collection and preparation of cells for isolation of total DNA from cultures and single cells, isolation of total DNA, PCR, qualitative assessment and purification of PCR products, processing and analysis of obtained sequences, phylogenetic analysis based on obtained gene sequences.

Exception: Nassonova Elena Stanislavovna performed full-genome amplification of DNA samples of Van79 and Tch1 isolates. The sequencing of the 18s rRNA gene of Thecamoeba striata CCAP 1583/4 strain used in the analysis was obtained by Nassonova Elena Stanislavovna and José Fahrny in 2001, but was mistaken by them for a contaminant sequence. Correctly identified and attributed, he was the author. Libraries were prepared and NGS sequencing and Sanger sequencing were performed at the Resource Centre of the St. Petersburg State University Science Park "Development of Molecular and Cellular Technologies". Purification and assembly of NGS sequencing data (isolates Van 79 and Tch1) was performed by Natalia Bondarenko and Mikhail Rayko, who also prepared the multigene alignment (isolate Tch1).

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2. Goal and tasks:

Goal: To study the diversity, systematics, and phylogeny of the amoebae of the family Thecamoebidae and to prepare material for a revision and monographic description of this family of amoebae.

Tasks of the work:

1. To isolate amoebae belonging to the family Thecamoebidae from natural habitats, establishing them in laboratory cultures if possible.

2. To study isolated strains by light microscopy, identify them or prepare materials for their description as a new species.

3. To amplify and sequence the 18s rRNA gene of isolated strains and performed a phylogenetic analysis.

4. To compare the results of phylogenetic analysis with data on the morphology of isolated strains. To assess the significance and reliability of morphological characters traditionally used for identification of amoebae of the family Thecamoebidae.

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3. Literature review

3.1 History of the study of the family Thecamoebidae and its current position in the lobose

amoebae system

Until the beginning of the twentieth century, most works on the systematics of amoeboid organisms presented general systems of "rhizopods", within which researchers were more interested in the general arrangement of groups of testate amoebae, foraminifera, and radiolarians (Leidy 1879; Butcschli 1882; Averintsev 1906; Penard 1902). In the early twentieth century, the individual more or less clearly families began to be established in the system of naked amoebae (Poche 1913; Schaeffer 1926).

In early articles, there are taxa under the names "Thecamoebida" and "Thecamoebina". These groups include testate amoebae and are contrasted with the groups "Gymnamoebida" and "Gymnamoebina", which include mainly naked amoebae (Averintsev 1906). However, in the further development of the amoeba classification these names have ceased to be used, and they are not the direct predecessors of the modern name of the family Thecamoebidae.

Schaeffer has developed a new system of naked amoebae. The system was based on an extensive analysis of known and new species of amoebae (Schaeffer 1926). The system was primarily based on the body form of the amoeba that it takes in locomotion. This idea of "locomotive form" as one of the most important traits of amoeba cell organization has survived to the present day and, in particular, has been used in works devoted to morphotypes of lobose amoebae (Smirnov and Goodkov 1999, Smirnov and Brown 2004). Schaeffer established the family Thecamoebidae in this new classification. The new taxon was located within the suborder Amreb^a Ehrenberg, 1830, order Lobosa Carpenter, 1861, class Rhizopoda Siebold, 1845. Schaffer comprises the genus Thecamoeba Fromentel, 1874 and a new genus Rugipes, established by him, to the family Thecamoebidae. The diagnosis of the family Thecamoebidae sensu Schaeffer 1926 was as follows: "These amoebae do not form pseudopods during locomotion. The shape in locomotion is ovoid to circular in outline and subject to slight change only. Longitudinal ridges in the ectoplasm are found in nearly all species" (Schaeffer 1926, p. 83).

Schaffer pointed out that amoebae of the genus Rugipes form long pseudopodia and take a "true ray form" "when disturbed", i.e. during flotation (from Page 1977). This trait is their main difference from thecamoebs. However, amoebae of these genera have no special differences in locomotive form when moving on the substratum. Schaeffer considered that the oval body shape and thick ectoplasm forming longitudinal ridges are main evolutionary trends of Thecamoebidae. Amoebae of the genus Rugipes are capable of forming discrete pseudopodia, although only during flotation,

therefore Schaeffer considered the genus Rugipes ancestral to Thecamoeba, whose amoebae never form pseudopodia. (Figure 1).

The new genus Rugipes comprised two species: R. vivax and R. bilzi. Subsequently, this genus was disbanded; its species were moved to other genera. R. vivax was included in a genus Vannella by Bovee in 1965, which was a part of subfamily Vannellina of family Flabellulidae (Bovee 1965). Then Page include R. vivax in a genus Platyamoeba, also included in subfamily Vannellina (Page 1969). Later, Sawyer established a new genus Clydonella within the family Thecamoebidae; and R. vivax was given the new name Clydonella vivax (Sawyer 1975). The second species - R. bilzi was transferred to the genus Thecamoeba in 1971 (Page 1971), but amoebae of this species have never been reisolated, so its taxonomic position remains unclear, and its validity remains questionable.

In the first half of the twentieth century, many articles proposing a variety of classifications of amoebae were published. All of them were based on different ways to analyse the features of amoeboid organisms, although generally oriented towards cell morphology (Kudo 1939; Raabe 1948; Hall 1953; Reichenov 1953). An attempt at a fundamentally new system was made by Singh (Singh 1952) and further developed by Chatton in 1953. Singh pays special attention to the characteristics of mitosis and, based on this, distinguishes new families. Further, Chatton established two new suborders: Mastigogenina ("with flagellate stage") and Amastigogenina ("without flagellate stage"), considering the presence of flagella as the most basal feature for the construction of the amoebae system. In this system, the family Thecamoebidae was included in the suborder Amastigogenina (Chatton 1953). Further development of the system based on features of mitosis eliminates almost all previously established families of amoebae. In 1970, Singh and Das proposed a new system of dividing the order Amoebida into only three families (Singh and Das 1970). Many genera of naked amoebae were not mentioned in the updated system, making it almost impossible to trace the fate of many species of the family Thecamoebidae.

Figure 1. Schemes and drawings from Schaeffer's work (from Schaeffer 1926).

А - Scheme of phylogenetic relationships of large amoebae. Б - Line drawings of consequential changes of locomotive form of naked lobose amoeba Thecamoeba rugosa (A), floating form (B) and "unusual" form of the cell in the old culture (C). В - Line drawings of consequential changes of locomotive form of naked lobose amoeba Rugipes vivax (A), various variants of the posterior end (B-D), outgrowths at the anterior end (E), outlines of the cell and the direction of cytoplasmic flow during a change in cell movement (F), and a scheme of the transition of the cell from the flotation to attached stage (G). Г - Locomotive form of Rugipes vivax. Д - Locomotive form of Thecamoeba hilla. Е - Locomotive form of Thecamoeba orbis. З - Locomotive form of Thecamoeba munda.

In the early part of the second half of the twentieth century, a series of articles by Bovee and Jahn once again substantially modified the amoeba system (Bovee 1954, 1970, 1972; Bovee and Jahn 1960, 1966; Jahn and Bovee 1965; Jahn et al. 1974). In Bovee and Jahn's 1966 system, the naked lobose amoebae were divided into three suborders within the superorder Lobida. The main trait of the division of these orders was the mechanism of amoeboid movement and features of cytoplasmic flow in cell pseudopodia. The family Thecamoebidae was the single taxon included in the suborder Rugina of the new order Thecida. In addition to the change of the position in the system, the composition of the family also changed. The genus Rugipes being transferred to sister family Striamoebidae. The diagnosis of the family Thecamoebidae coincided with that of the suborder and was as follows: "Large amebas with heavy, much-wrinkled ectoplasm in locomotion; wrinkles dorsal, more or less parallel; pseudopods only as blunt bulges" (Bovee and Jahn 1966, p. 233; Jahn et al. 1974, p. 430).

The composition of the genus Thecamoeba was also greatly modified: many species previously included in it were included in the new genus Striamoeba of the family Striamoebidae, suborder Striatina. The suborder Striatina as well as the suborder Rugina belonged to the suborder Thecida. The diagnosis of the family Striamoebidae, identical to that of the suborder Striatina, was: "Small amebas with distinct parallel dorsal longitudinal ridges in locomotion; pseudopods rarely formed, then only as blunt bulges". A similar separation of groups of amoebae "large with wrinkles" and "small with ridges" also found its way into the modern classification of locomotive forms and corresponds to the rugose and striate morphotypes, but the classification of morphotypes does not take into account the size characteristics. Bovee and Jahn did not specify a clear boundary between "large" and "small" amoebae in the new system; the 100 |im boundary appeared in a more recent article, which is a key of amoebae compiled by Bovee (1985). Thus, with all new and extant old taxa in these works, species of the family Thecamoebidae have been assigned to different groups within the order Thecida (Fig. 2).

Figure 2. Line drawing of thecamoebids (from Bovee and Sawyer 1979).

A - Floating and locomotive forms of "Thecamoeba terricola (verrucosa)". E - Floating and locomotive forms of "Striamoeba hilla". B - Floating and locomotive forms of "Striamoeba rugosa". r - Floating and locomotive forms of "Striamoeba orbis". ^ - Locomotive forms of "Striamoeba munda".

Scale bar: A - 100 ^m, E-fl - 50 ^m.

The next stage in the evolution of the amoeba system was Page's 1976 work (Page 1976a). Page established Thecida as a suborder within the order Amoebida. The family Thecamoebidae became single in the taxon Thecidae and its composition was substantially changed: the genera Thecamoeba, Sappinia, Platyamoeba, Pessonella and Vannella were included in the family. Species of the genus Thecamoeba, previously transferred to the Striamoebidae family, were returned. Page included the genus Sappinia after publication of a paper on the morphology and ultrastructure of Sappinia diploidea (Goodfellow et al. 1974). The genus Rugipes had already been disbanded and did not appear in this new system. Also, Page includes the genera Vannella Bovee, 1965 and Platyamoeba Page, 1969, which were previously considered related to the genus Flabellula. One more new genus, included by Page into the family Thecamoebidae, is Pessonella Pussard, 1973, which like Vannella with its fan-shaped body, but has "papilla-like" outgrowths on the surface of the frontal hyaloplasm zone (Pussard 1973). Amoebae of Pessonella have never been reliably reisolated subsequently. Thus, Page included all naked lobose amoebae in the family Thecamoebidae, which have smooth rounded outlines and which lack pseudopodia during locomotion. Page's 1976 proposed diagnosis of the family is similar to proposed by him for the suborder Thecida: "Flattened, often oblong, ovate, or flabellate and more or less regular in outline; often with extensive hyaline zone; often with discernible pelliclelike layer which may be distinctly wrinkled; rolling movement of surface in locomotion, with most species never-showing any branching; nuclear division patterns diverse" (Page 1976a, p.67).

Page's further work on the reclassification of the lobose amoebae again led to significant changes in the position and composition of the family (Fig. 3). These changes followed a series of articles about the ultrastructure of amoebae. The family Thecamoebidae was transferred to the taxon Euamoebida Lep§i, 1960, which became an order at Page's suggestion (Page 1987). The genera Platyamoeba, Pessonella and Vannella were excluded from the family. Specifically, the genus Vannella was returned to the family Vannellidae, as in Bovee's 1970 article; the genus Platyamoeba was moved there as well. This was due to the discovery of penta- or hexagonal glycostyles typical for the Vannellidae (Page and Blakey 1979). Also, two new genera: Dermamoeba Page et Blakey, 1979 and Thecochaos Page, 1981, were included in the family Thecamoebidae. Amoebae of the genus Dermamoeba have smooth outlines without pseudopods and rarely form folds on the body surface. A significant feature is the presence of a thick, bilayered, complex glycocalyx, in contrast to many other thecamoebids, which have an amorphous glycocalyx. Page established the genus Thecochaos only on the basis of permanent preparations made by Penard (Page 1981). Page included these amoebae to the Thecamoebidae, despite the fact that the cells had a lobed body shape and many nuclei, which is not typical for other Thecamoebidae. The family Thecamoebidae in this work included the genera: Thecamoeba, Sappinia, Dermamoeba, and Thecochaos. Page gives a new version of the diagnosis of the family Thecamoebidae: "Flattened, with more or less regular outline, usually oblong, monopodial

with rare exceptions, often with discernible pellicle-like layer, which may be distinctly wrinkled: hyaloplasm usually a deep, anterolateral crescent. Usually uninucleate; one genus binucleate and one multinucleate; nucleolar material in few or many parietal pieces or one or two central pieces. No cytoplasmic crystals. Surface: covered with thick, amorphous glycocalyx or, in one genus, a cuticle. Distinct inner fibrous nuclear laminae known in some species." (Page 1987, p. 205).

In 1988, Page established a new genus Pseudothecamoeba within the family Thecamoebidae, created for the peculiar species Thecamoeba proteoides Page, 1977, which he had previously described. These unusual amoebae have an elongated shape and have folds on the posterior half of the cell during locomotion. One of them main features is the large number of vacuoles in the cytoplasm. When moving slowly, Pseudothecamoeba can form individual pseudopodia, making the cell outline similar to that of proteus amoebae (Page 1988).

Figure 3. Diversity of the family Thecamoebidae sensu Page 1988.

А - Thecamoeba striata (from Page 1988). Б - Thecamoeba terricola (from Page 1988). В -Sappinia diploidea (from Page 1988). Г - Dermamoeba granifera (from Page 1988). Д - Thecochaos album (from Page 1981). Е - Pseudothecamoeba proteoides (from Page 1988).

In 1993, Rogerson established the genus Parvamoeba, which he originally placed in the family Thecamoebidae (Rogerson 1993). Amoebae of the new genus have a rounded or slightly elongated outline in locomotion, the cell surface has an irregular wrinkled and is covered by a thick amorphous glycocalyx. However, further studies of this peculiar organism have led to its transfer into the family Parvamoebidae, order Himatismenida Page, 1987 (Kudryavtsev 2012; Tekle et al. 2016).

In the same year, Smirnov and Goodkov (1993) established the genus Paradermamoeba within the family. Amoebae of this genus have an elongated lanceolate body with lateral lobes and do not form discrete pseudopodia during locomotion. Another feature of these amoebae is the thick glycocalyx consisting of spirally twisted glycostyles, unique among the lobose amoebae. In the same paper, the authors criticized the diagnosis of the family Thecamoebidae. They stated that the listing of types of nucleus organization and the emphasis on the glycocalyx organisation, which is extremely diverse among thecamoebids, are rather vague traits and should be removed from the family diagnosis in the future. A year later, in a 1994 article (Smirnov and Goodkov 1994), the same authors provided a corrected diagnosis of the family Thecamoebidae: "Amoebae flattened, with more or less regular outline, commonly oblong, monopodial with rare exceptions; at light-microscopical level surface often appears as pellicle-like layer, which may be distinctly wrinkled; hyaloplasm usually a deep, anterolateral crescent, sometimes with long lateral extensions toward the posterior end, but continuous anterior hyaloplasm never occupies up to half of body length; a variety of nuclear structures; no cytoplasmic crystals." (Smirnov and Goodkov 1994, p. 113).

The family has undergone further changes following a series of molecular studies (Peglar et al. 2003; Smirnov et al. 2005; 2007; 2011). On the basis of morphological and molecular data, the genera Dermamoeba and Paradermamoeba were transferred from the family Thecamoebidae into a separate family Dermamoebidae. Also, according to the results of phylogenetic analysis, the genus Stenamoeba, containing species previously included in the genus Platyamoeba of the family Vannellidae, was included in the family. The genera Thecochaos and Pseudothecamoeba were assigned the status Lobosa incertae sedis due to their strong distinction from all known groups of lobose amoebae and lack of current data. The diagnosis of the family Thecamoebidae was identical to that of the order Thecamoebida: "Amoebae with smooth outlines, oblong, striate or rugose, with deep anterolateral hyaline crescent. Cell surface wrinkled, often with longitudinal dorsal folds. Cell coat thin, dense, amorphous or its basal layer is amorphous" (Smirnov et al. 2011, p. 565).

The number of Thecamoebidae genera has undergone recent changes in the work of Melton et al. (Melton et al. 2019). The organisms studied were morphologically defined as thecamoebids with a broad locomotive form and many transverse folds, indicating their possible belonging to the rugose morphotype. However, the presence of short lobate pseudopodia strongly distinguishes them from all known thecamoebids. The assumption that the new amoebae are a new genus was also confirmed by

the results of phylogenetic analysis based on multigene alignment. A new genus Stratorugosa has been established based on the results of a study by Melton with co-authors (Melton et al. 2019).

Further molecular studies have brought no new changes to the number of genera and position of the family Thecamoebidae (Fig. 4). In the present work, we use the system of lobose amoebae proposed in Adl et al. (Adl et al. 2019). The composition and history of each of the genera reliably belonging to the family Thecamoebidae as well as the genera Pseudothecamoeba and Thecochaos are observed in the next chapter.

Figure 4. The modern diversity of Thecamoebidae.

А - Thecamoeba quadrilineata (from Bondarenko et al. 2022). Б - Thecamoeba aesculea (from Kudryavtsev and Hausmann 2009). В - Sappiniaplatani (from Wylezich et al. 2015). Г and Д -Sappinia pedata (from Brown et al. 2007). Е - Sappinia sp. strain SG10G (from Tyml and Dykovâ 2018). Ж - Stenamoeba berchidia (from Geisen et al. 2014). З and И - Stenamoebapolymorpha (from Peglar et al. 2016). К-Н - Stratorugosa tubuloviscum (from Melton et al. 2019). Scale bar: А-Г, Е-И - 10 |m, Д - 100 |m, К-Н - 20 |m.

3.2 Genera of the family Thecamoebidae

Genus Thecamoeba Fromentel, 1874

The genus Thecamoeba includes naked lobose amoebae of the rugose and striate morphotypes (Smirnov and Goodkov 1999; Smirnov and Brown 2004). Thecamoeba are relatively easy to identify in culture due to the folds and ridges on the cell surface and their smooth outline during locomotion. A modern diagnosis of the genus Thecamoeba is given in Page's key: "A flattened ovoid or oblong, with length usually less than 2.5 times width; usually with longitudinal surface wrinkles, or folds; hyaloplasm an anterior crescent, often with lateral extensions toward posterior end; not branching. Normally mononuclear. A thick, dense, commonly amorphous glycocalyx. No cysts known" (Page 1988, p. 68).

The genus Thecamoeba was established by Fromentel in 1874 (Fromentel 1874, p. 222). Fromentel included in the new genus one species - T. quadripartita, which is considered a type species of the genus Thecamoeba. Describing Thecamoeba quadripartita, he spoke of an "amoeba in armour" which "is divided into four longitudinal parts, and each part moves along a dividing line as on a hinge" (Fromentel 1874, p. 346). The illustration shows an amoeba with a drop-shaped outline (Fromentel 1874, Plate 28, fig. 3). There are three "sutures', which are probably longitudinal ridges. The cytoplasm is divided into granuloplasm and a frontal layer of hyaloplasm, which continues along the lateral sides of the cell to the posterior end. In the granuloplasm, only a large contractile vacuole is among the signed structures. In the granuloplasm there is a spindle-shaped structure that is most likely the contents of the digestive vacuole. However, from Fromentel's descriptions, it is difficult to establish the morphological traits of T. quadripartita that would help to identify this species within the modern system of the genus Thecamoeba. In particular, the description or the illustration doesn't demonstrate the morphology of the nucleus - one of the most important traits for the identification of Thecamoeba; there is also no data about the cell size of T. quadripartita. Because of the lack of morphological data, Page, does not express any hope that a type species for Thecamoeba will ever be reliably reisolated and described in detail (Page 1977).

The names of some species, which now belong to the genus Thecamoeba, predate the work of Fromentel, such as Amoeba verrucosa Ehrenberg, 1838. The number of species now included in the genus Thecamoeba increased after the series of works by Greeff (Greeff 1866, 1891) and Penard (Penard 1902, 1905, 1913) on the study of "amoebae with pellicle". The species described by these authors were placed in the genus Amoeba. Schaeffer adds new species directly to the genus Thecamoeba in a paper that established the family Thecamoebidae (Schaeffer 1926). He also moves the previously described species Amoeba (=Thecamoeba) sphaeronucleolus Greeff, 1891, Amoeba (=Thecamoeba) vesiculata Penard, 1902 and Amoeba (=Thecamoeba) striata Penard, 1890 into the

genus Thecamoeba. Later, in 1960, three more species T. quadrilineata, T. similis and T. terricola (Lep§i 1960) were transferred to the genus.

According to Page's 1971 and 1977 papers, which are essentially a monograph about the genus Thecamoeba, 18 known species are may belong to thecamoebs (Table 1). Page also points out that he does not mention some "species" in his works, attributing this to the fact that their description is questionable, and thinks that these names should not be used (Page 1971, 1977). Nowadays, the genus Thecamoeba includes 9 species for which detailed descriptions and illustrations are available (Page 1991, 1977; Smirnov 1999a; Kudryavtsev and Hausmann 2009). These species can be relatively easily identified as thecamoebs, and they can be distinguished. More species can be found in the literature that are formally named Thecamoeba and potentially belong to this genus. However, by the modern system, descriptions of these species lack the data necessary to reliably assign them to the genus Thecamoeba, and some species are almost impossible to recognize from available descriptions, even if they are found again (Page 1977, 1991).

Several attempts have been made to establish separate groups of species within the genus Thecamoeba during the study of it. One attempt to divide the genus Thecamoeba was described above: in the 1966 classification of Bovee and Jahn, not even the genus was divided into subgroups, but Thecamoeba species were distributed between two suborders of the order Thecida. Based on the suborder diagnoses, the main feature for division was the size characteristics and morphology of the locomotive form. One group included large amoebae (over 100 |im), forming many multidirectional wrinkles during movement and belonging to the rugose morphotype according to the modern classification. The second group includes small amoebae (less than 100 |im) forming dorsal longitudinal folds parallel to the course of movement and thus belonging to the amoebae of the striatal morphotype. A similar concept has been proposed by Page (1977): the genus Thecamoeba can be divided into three groups based on the morphology of the locomotive form. The first "verrucosa-like" group comprises amoebae whose surface is covered with many multidirectional folds and wrinkles during movement. The name of this group is similar to the species epithet Thecamoeba verrucosa, which Page includes in the group. A second group, "striata-like", takes its name from Thecamoeba striata, similarly. The second group includes species whose species have smooth outlines, have practically no wrinkles, and whose dorsal surface often has several longitudinal folds during locomotion. The third group includes only one species, T. proteoides, capable of forming discrete pseudopodia. This species was later transferred to the separate genus Pseudothecamoeba (Page 1988).

Another version of the division of the genus Thecamoeba into groups was proposed by Pussard (Pussard 1973). In his opinion, genera of amoebae with different mitotic patterns cannot belong to the same family. Also, a genus should include morphologically similar species. On these rules, Pussard concludes that the genus Thecamoeba is in complete contradiction to the rule of homogeneity and the

genus is needed to be split. Page also notes a difference in the mitotic patterns of different species of Thecamoeba, but does not divide the genus based on this feature. In 1982, Singh et al. proposed to divide the genus Thecamoeba into 8 independent genera based on the pattern of cell mitosis and nucleus structure (Singh and Hamemaiah 1979; Singh et al. 1982), but this proposal was not supported by the work of other researchers.

Molecular data for the genus Thecamoeba remain incomplete. GenBank contains sequences from three of the nine species described in detail - T. similis, T. aesculea and T. quadrilineata. There are also sequences labelled as Thecamoeba sp. but actually representing other groups of Amoebozoa. "Thecamoeba sp. ATCC PRA-35" refers to Parvamoeba monoura (Cole et al. 2010) and "Thecamoeba sp. JRG-2011 ATCC 50185" is a Sappinia strain (Corsaro et al. 2017). There are also transcriptome data on Thecamoeba quadrilineata ATCC PRA-259, which by origin is strain CCAP 1583/10 (Tekle et al. 2016), and data on the unidentified strain "Thecamoeba sp. SK13-4B" (Kang et al. 2017).

Phylogenetic analysis based on 18s rRNA gene sequences clearly shows that Thecamoebidae are a distinct monophyletic taxon within the family Thecamoebidae (Smirnov et al. 2011).

Table 1. Species of the genus Thecamoeba

Described in detail, valid species:

1. Thecamoeba aesculea Kudryavtsev et Hausmann, 2009

2. Thecamoeba hilla Schaeffer, 1926

3. Thecamoeba munda Schaeffer, 1926

4. Thecamoeba orbis Schaeffer, 1926

5. Thecamoeba quadrilineata (Carter, 1856) Lep§i, 1960

6. Thecamoeba similis (Greeff, 1891) Lep§i, 1960

7. Thecamoeba sphaeronucleolus (Greeff, 1891) Schaeffer, 1926

8. Thecamoeba striata (Penard, 1890) Schaeffer, 1926

9. Thecamoeba terricola (Greeff, 1866) Lep§i, 1960

Candidate species for a position within the genus Thecamoeba:

1. Thecamoebapapyracea (Penard, 1905) Lep§i, 1960

2. Thecamoeba rugosa Schaeffer, 1926

3. Thecamoeba verrucosa (Ehrenberg, 1838) Schaeffer, 1926

4. "Striamoeba" sparolata Fishbeck et Bovee, 1993

Types of questionable validity:

1. Thecamoeba assimilis Lep§i, 1960

2. Thecamoeba bilzi (Schaeffer, 1926) Page, 1969

3. Thecamoeba corrugata Bovee, 1953

4. Thecamoeba hoffmani Sawyer Hnath et Conrad, 1974

5. Thecamoeba quadripartita Fromentel, 1874

6. Thecamoeba ovalis Lep§i, 1960

7. Thecamoebapulchra (Biernacka, 1963) Page, 1977

8. "Greeffia" soli Singh et Hanumaiah, 1979

9. "Sahnium" lucknowensis Singh, Misra et Sharma, 1982

10. "Carteria bengaliensis Singh, Misra et Sharma, 1982

T. aesculea Kudryavtsev et Hausmann, 2009

The species T. aesculea was described in 2009 from an amoeba culture isolated from the bark of the Aesculus hippocastanum. The amoebae have an anteriorly expanded, oval, rounded to fan-shaped outline with a slightly retracted posterior end. The locomotive cells range in length from 70 to 119 |im (101.5 |im on average); width from 56 to 118 |im (80.4 |im on average), with a length to width ratio of 0.89 to 1.93 (1.29 on average). The surface is covered by many wrinkles, and three to five irregular longitudinal folds may be present on the dorsal surface. The combination of traits of the locomotive form allows these amoebae to be belonged to the rugose morphotype. The large nucleus has a single centrally located nucleolus with circular outlines. A clearly visible layer of fibrils, about 0.3 |im thick, is located inside the inner nuclear membrane. On microphotographs, the nucleolus also has clear circular outlines. The texture of the nucleolus is loose, but noticeably darker than the texture of the rest of the nucleolus. The outer layer is loose but homogeneous, while the central part of the nucleolus is divided into dense dark and light areas with a clear boundary between them. The cell surface has an amorphous glycocalyx up to 33 nm thick. A basal electron-dense layer 8-10 nm thick and a less dense outer layer about 10-15 nm thick, separated from the basal layer by an electron-light space 8-10 nm thick, can be distinguished in the glycocalyx. The type culture was deposited in CCAP under number 1583/12, but has now been lost.

GenBank has one 18s rRNA gene sequence of T. aesculea, which was obtained as part of the work on the revision of the naked lobose amoeba system in 2011 (Smirnov et al. 2011). The sequence has a 432-nucleotide long insertion in the helix 31 (by Petrov et al. 2014), which is conserved for Thecamoebidae. Phylogenetic analysis shows a sister species arrangement of T. aesculea and T. quadrilineata. Both species have a vesicular nucleus.