Turkey.

University of Jaén Faculty of Experimental Sciences Department of Animal Biology, Plant Biology and Ecology

Academic year 2013 – 2014

Diversity and distribution of soil nematodes in Mount Ararat (5137 m), Turkey. Taylan Çakmak

Taylan Çakmak

Promoters: Prof. Dr. Reyes Peña-Santiago Prof. Dr. Wim Bert Thesis submitted to obtain the degree of European Master of Science in Nematology

ACKNOWLEDGEMENTS “Almost there! Keep going Taylan! Almost there!” These are the words that I was telling myself when we were in the last iced slope at the top of Mount Ararat. It was excessively cold but much more happiness for me. Honestly, this expedition of Mount Ararat was the top of my short career and it was literally a lifetime experience. There are so many people who paved the way to make this experience real and the names must be mentioned here. Especially, I would like to thank firstly to Nic, Professor Decreamer and Inge who are the organisers of EUMAINE program for bringing me this special opportunity as being a part of the nematology family and of course for the financial support. Secondly of course my Promoter Professors; Prof. Dr. Reyes PeñaSantiago; I cannot imagine finishing this work without his comprehensive helps and advices, very special thanks go to him for always being helpful. It was another valuable chance for me to learn his way of working. Prof. Dr. Wim Bert; I remember when he encouraged me to do this project, once he asked me: “Why don’t you take this subject?” Yes Wim, we did it together! You encouraged and supported me at every steps of my thesis work with a great patience and also thanks for the financial support on the project. So many thanks go to both of them! Also my previous promoter, Assoc. Prof. Dr. Ugur Gozel from Turkey, my special thanks go to him for putting me into nematology world and letting me work in his lab anytime I want. Indeed everything started in that laboratory with his priceless advices! Prof. Pablo Castillo, thanks to him, I have gained a remarkable knowledge about genus Rotylenchus. Thank you Professor Castillo! Dr. Abolafia, we could manage to identify this challenging group of nematodes, Rhabditids with him. Thank you very much for your valuable help! Dr. Sergio Álvarez-Ortega, thank you very much for your help on the group Aporcelaimidae, it was much appreciated. Prof. José A. Carreira (Pepe) thank you very much for your help on statistical analyses, he is wellskilled in soil ecology. I also want to thank all the professors who taught me during my stay at Gent University and at Jaén University. I have learnt a lot of new things, and now I am feeling “ready” for the world of nematodes! A part of the laboratory work has been done in Turkey, so I must mention here my thanks to Seda Musdagi for being my dear nematology friend and helping me on the extraction of nematodes. Dr. Ali Sungur, he is our elder brother (“Abi” in Turkish) thank you abi for helping me on soil moisture analyses. And also of course here I must mentioned my mounteneers, super kind, super fine friends; 15 young scientists from five different countries; Oriol Rios, Bernat Rios, Euldald Correig Fraga, Oriol Parera, Ferran Gibert, ii

Maria Munoz Cervantes, Maria Dalmaces, Jonathan Strick, Baykal Onal, Adonis Kopsacheilis, Eloi Vilamajó Llobera, Joan Atcher, Xavier Clotet, Emrah Avci and our superguide Sadik Yildiz. This work could not have been accomplished without the valuable help of these nice people. Also my EUMAINE collegues, we spent a priceless year 2013 together and I will definitely visit most of you in some days. It was a great pleasure to meet all of you. Especially I would like to thank Sevgi Turkoz and Israel Ikoyi who helped me a lot last year during the adaptation period of studies and also Amjad Zia for his help on various steps of my thesis. And finally special brakets must be opened for my family. Thank you very much! They always stand by me and they are “the breath of life” for me.

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Diversity and distribution of soil nematodes in Mount Ararat (5137 m), Turkey Taylan ÇAKMAK Departamento de Biología Animal, Vegetal y Ecología, Universidad de Jaén, Campus “Las Lagunillas” s/n, Edificio B3, 23071- Jaén, Spain.

Summary - The diversity and distribution of soil nematodes was studied in Mount Ararat (5137 m) from an altitude of 1523 m to 5000 m a.s.l. along five different eco-habitats. A total number of 2560 nematodes were identified belonging to 31 families, 62 genera and 70 species. Fortyfour taxa were firstly recorded for Turkey`s nematofauna and 27 species are morphologically and/or morphometrically characterized, including scanning electron microscope (SEM) pictures of Acrolobus longigubernaculum, Cervidellus vexilliger, Rotylenchus sp. and Tylocephalus auriculatus. Additionally, molecular analysis of D2–D3 expansion of 28S rRNA genes were provided for three species, namely Heterodera trifolii, Pratylenchus thornei and Rotylenchus sp., and ITS1-rRNA gene sequences were obtained for Rotylenchus sp. Both morphological and molecular evidences support that the material herein identified as Rotylenchus sp. certainly belongs to a non-described species of this genus, very close to R. conicaudatus. The nematode diversity (species richness) and the nematode abundance display recognizable patterns of altitudinal distribution as the number of species reaches a maximum at intermediate elevations, whereas the nematode abundance is significantly higher at the highest altitudes. The nematode community associated to marshland habitat becomes significantly different from those associated to the other four eco-habitat types. The mean value of the maturity indices were similar in all studied sampling sites and did not differ significantly with the altitudinal gradient or eco-habitat type. Overall, bacterivorous nematodes were the most abundant group and dominating the community in all sampling sites (52%). Keywords Altitude, community analysis, distribution patterns, morphology, Nematoda, phylogeny, taxonomy.

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Diversidad y distribución de los nematodos edáficos del Monte Ararat (5137 m), Turquía

Resumen – En este trabajo se estudia la diversidad y la distribución de los nematodos edáficos del Monte Ararat (5137 m) en un rango altitudinal que va desde 1523 m hasta 5000 m s. n. m. y en cinco eco-hábitats diferentes. Se han identificado un total de 2560 nematodos pertenecientes a 31 familias, 62 géneros y 70 especies. Cuarenta y cuatro taxones se citan por primera vez para la nematofauna de Turquía y 27 especies se caracterizan morfológica y morfométricamente, incluyendo

estudios

de

microscopía

electrónica

de

barrido

(MEB)

de

Acrolobus

longigubernaculum, Cervidellus vexilliger, Rotylenchus sp. y Tylocephalus auriculatus. Además, se presenta un análisis molecular de la expansion D2–D3 del gen ARNr-28S para tres especies, a saber Heterodera trifolii, Pratylenchus thornei y Rotylenchus sp., así como secuencias del gen ARNr-ITS1 para Rotylenchus sp. Tanto las evidencias morfológicas como las moleculares sostienen que el material identificado como Rotylenchus sp. pertenece con toda certeza a una especie no descrita de este género, muy próxima a R. conicaudatus. La diversidad (riqueza específica) y la abundancia nematológicas muestran patrones reconocibles de distribución altitudinal puesto que el número de especies alcanza un máximo en altitudes intermedias en tanto que la abundancia es significativamente mayor en las altitudes más elevadas. La comunidad nematológica asociada a áreas pantanosas resulta ser significativamente diferente de las asociadas a otros tipos de eco-habitats. El valor medio de los índices de madurez son similares en todos los lugares muestreados y no difieren significativamente respecto al gradiente altitudinal o al tipo de eco-hábitat. En conjunto, los nematodos bacteriófagos son el grupo más abundante y dominante de la comunidad en todos los lugares estudiados (52%). Palabras clave Análisis de comunidad, filogenia, gradiente altitudinal, morfología, nematodos, patrones de distribución, taxonomía.

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Various biogeographic reasons explain the unique diversity met in Turkey`s nature. Due to its “crossroads” location and the variations of its geographic features and climatic conditions, Turkey hosts a rich biodiversity. Another factor that has shaped Turkey`s biodiversity is the ice ages ranging from 1.8 millions to ten thousand years ago. The global map of “biodiversity hotspots” clearly reflects this richness. Three out of 34 “biodiversity hotpots” meet in Turkey and one of them is the Irano-Anatolian spot. About one-fifth of the world’s land surface area is occupied by mountain areas and 11% of the terrestrial surface lies above 2000 m a.s.l. Mountains are very special places of our Earth, with their extreme habitats and fragile ecosystems, which are adapted to excessive rainfall, low temperatures and intensive solar radiation. Many different factors can play role associated to species richness, distribution patterns and altitude. The two most important factors which are globally associated with altitudinal gradient are air temperature decline and the decrease of terrestrial area per bioclimatic belt (Körner, 2007). These factors have a direct impact on the mechanisms of adaptation related to temperature, mountain geometry and the age of the mountain (Körner et al., 1991). Previous studies in which nematodes have been studied associated to mountains were based on taxonomical and ecological approaches (Hoschitz & Kaufmann, 2004). Nevertheless, there are very few studies associated to altitudinal distribution of nematode assemblages (Smolik & Weaver, 1987; Chen et al., 2009; Háněl & Čerevková, 2010). Basically, these studies were conducted on temperate and subtropical forests. Therefore, the association concerning nematode diversity and altitudinal gradient in high mountain areas rather than forests environments is still not very well known. Mount Ararat is located at the coordinates 39°42.113′N and 44°17.899′E, in Eastern Region of Turkey, between the provinces of Doğubayazıt and Iğdır, near the border of Iran, Armenia and Nahcivan. It is the highest peak in Anatolia with an altitude of 5.137 m or 16.854 ft and a 400 m diameter permanent ice cap in the summit. The Mountain is a type of stratovolcano, also known as a conical volcano, made of many layers (strata) of hardened lava, tephra, pumice and volcanic structure. It erupted in 1840 for last time. Unlike shield volcanoes, stratovolcanoes are characterized by a steep profile and periodic explosive eruptions and quiet eruptions (IBS, 1964). The Mountain is located in the most thinly populated region of the country. Farming activities are difficult because of long, severe winters, steep slopes and eroded soil. However 3

grain, chiefly summer wheat and barley, are grown in foothills. There are pastoral nomads in lower parts of the Mountain who raise sheep and goats (IBS, 1964). Due to harsh and long winter periods, flora and fauna drastically changes in different altitudes.

Fig. 1. Panoramic view of Mount Ararat with altitudinal gradient. Depending on geographical site of Month Ararat, mainly flora is located in between 21003800 m, being dominated by grasslands. After this point, most of the plants cannot adapt to the climate and only some mountain wildflower meadows can rarely be found. Many endemic species of flora were recently found at the foothills of Mount Ararat (Koyuncu, 2005). Nevertheless, studies focusing on the distribution, ecology and diversity of terrestrial nematodes in Mount Ararat are completely absent. Therefore Mount Ararat was chosen as a research site.

Objectives The general aim of this contribution is to explore the nematode fauna dwelling the soils of Mount Ararat, a pristine territory for nematological studies. More specifically, the following objectives are pursued: i) to characterize the soil nematode fauna, 4

ii) to describe the altitudinal distribution of the species, iii) to detect tentative distributional patterns of nematode diversity, and iv) to study the distribution of nematode assemblages.

Materials and methods

SAMPLING Samples were collected at Mt. Ararat, Turkey, in July 2013 during the expedition of the project “Worms4Ararat” organized by a Turkish and a Catalonian hiking clubs with 15 young scientists from six different countries. More information about the project can be found on the website http://worms4ararat.webnode.es/. The sampling was done regarding to an altitudinal gradient and along five different eco-habitats, namely wildflower meadow, mountain grassland, chalk grassland, riverbed and marshland (Fig. 2). Samples were collected from 30 sampling sites. For each location, one soil sample were collected from 15 x 15 cm plot and from one location an ice sample was collected from 30 cm depth. A total number of 29 soil samples and an ice sample were put into ziplock sampling bags, stored in portable cooler during the transportation and brought to nematology laboratory for extraction process.

EXTRACTION Nematodes were extracted by using modified Baermann’s (1917) funnel technique. After separating rocks and big organic particles, further processing was done based on improving the uniformity of samples within an equal 200 ml volume of bakers. For each soil sample, a volume of 200 cc was used. Samples were placed on plastic trays lined with paper towels and incubated on the laboratory. Nematodes were collected after 48 hours by pouring the extraction tray over a 500 mesh sieve (25 μm opening) and put into DESS solution according to Yoder et al. (2006). Each extract was then labeled with corresponding sample number and transported in plastic tubes to the University of Jaén, Spain. Remaining samples were also brought to the laboratory as backup for further studies.

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Fig. 2. Vertical distribution of sampling sites and corresponding eco-habitats in Mount Ararat.

DETERMINATION OF SOIL MOISTURE Calculation of the soil moisture was simply based on the dry matter content of each sample. A 20 g sub-sample was taken from each moist soil sample and weigh. Sub-samples were then dried at 105°C for 24 hours until the weight does not decrease any further (Southey, 1986). Then the weight for each sub-sample was measured after drying, divided by the weight before and calculated the soil moisture content.

RECOVERY OF ENTOMOPATHOGENIC NEMATODES A 100 g soil sample from each sampling sites was placed into glass container each with three last instar larvae of the wax Moth Galleria mellonella (L.) and covered with a lid. (Bedding & Akhurst, 1975; Kaya & Stock, 1997). The samples were then stored at room temperature.

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After 10 days, dead larvae were collected and transferred to White traps to collect the emerging IJs (Kaya & Stock, 1997).

PREPARATION OF NEMATODES FOR LIGHT MICROSCOPE After picking up procedure, all the preserved nematodes were rinsed with purified water to remove the debris. The staining block with extracted nematodes were then placed in an airtight jar which contains 1.25 cm deep volume of 96% ethanol, added a few drops of glycerol:formaline (4%) (1:99) and left overnight at room temperature. Next morning, the staining block was removed from the jar and added few drops of five parts glycerol and 95 parts 96% ethanol solution, covered two thirds of its cavity with a glass square and placed in an incubator at 40°C. For gradual transition of glycerin, a few drops of glycerol:ethanol (5:95) solution was added every two hours. The day after, individual nematodes were permanently mounted on glass slides (Yoder et al., 2006).

SCANNING ELECTRON MICROSCOPY Fixed specimens were rinsed into deionized water to remove all traces. After two hours for dehydration of specimens, nematodes were placed into ethanol concentration by passing them through series of 25% (overnight), 30%, 50%, 70%, 80%, 90%, 95%, 100%, 100% (each for two hours). The day after specimens were put in acetone for 60 minutes before critical point dried with CO2, coated with gold (Abolafia & Peña-Santiago, 2005) and observed with a Jeol JSM5800 microscope. MEASUREMENTS Measurements and drawings were made manually by using a drawing tube attached to an Olympus microscope (Olympus Optical, Tokyo, Japan). Light microscope pictures were taken by using a Nikon Eclipse 80i microscope equipped with a Nikon Digital Sight DS-5M digital video camera. Illustrations were prepared based on light microscope drawings by using Illustrator® CS3 Extended software, version 10.0 (Adobe Systems, CA, USA).

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MOLECULAR CHARACTERIZATION

DNA Extraction, PCR and Sequencing DNA from Rotylenchus, Pratylenchus and Heterodera species were extracted from single individuals. After initial selection, nematodes were picked up from the DESS solution, rinsed with deionized water, put in a drop of worm lyses buffer (50 mM KCl; 10 mM Tris-Cl pH 8.3; 2.5 mM MgCl2; 0.45% NP 40; 0.45% Tween 20), cut into small pieces and after adding 60μg ml−1 Proteinase K, nematodes were taken to a micro centrifuge tube and incubated at −80 ℃ for 10 min. DNA suspension was added to PCR mixture after preparation the tubes by incubating for 1 hour at 65℃ followed by enzyme deactivation for 10 minutes at 90℃ and centrifugation 1 minute at 16,000g. PCR was performed with a final volume of 25μL suspension, containing 2.5μL genomic DNA template, 2.5μL of 10× reaction buffer with MgCl2, dNTP-mix at 0.2 mM each, 0.5 m of each primers; D2Ab (5´-ACAAGTACCGTGAGGGAAAGTTG-3´), D3B (5´TCGGAAGGAACCAGCTACTA-3´) for the D2-D3 domain of the large subunit (LSU) rDNA gene (De Ley et al., 1999) and Vrain2 forward (5´-CTTTGTACACACCGCCCGTCGCT-3´), Vrain2 reverse (5´-TTTCACTCGCCGTTACTAAGGGAATC-3´) for the ITS1-rRNA (Vrain et al., 1992). The PCR amplification was performed with initial denaturation at 94℃ for 5 min, followed by 35 cycles of 94℃ for 1 min, 54℃ for 90 s and 72℃ for 2 min, and a final extension at 72℃ for 10 min. PCR products were sized with 1 kb DNA ladder (Promega, Madison, WI, USA) on a 1% agarose gel stained with 0.0003% ethidium bromide. In order to obtain sequences of the forward and reverse DNA strand, PCR products were sent to Macrogen Inc, Netherlands and sequencing was done in both directions with the appropriate primers. The sequences were then edited by performing BioEdit 7.0.4.1 (Hall 1999). The obtained sequences were aligned together with available sequences from Genbank (NCBI)

Phylogenetic analyses D2-D3 domains of 28S and ITS1-rRNA were used for phylogenetic analyses. Previously published sequences were obtained from GenBank to perform phylogenetic reconstruction. Alignment of sequences were checked by using program CLUSTAL W (Thompson et al., 1994). Bayesian phylogenetic inference (BI) was performed with MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003). The best fitting model with rate variation across sites and a proportion of 8

invariable sites (GTR + I + G) as estimated by PAUP/MrModeltest 1.0b (Nylander, 2004) was performed for entire alignment. Two independent runs were performed for each 3 million generations and the trees were obtained by using the last 1 million generations with well beyond the burn-in value. Final statistics to check two different runs as average standard deviation of split frequencies between the two analyses were convincing as the value approached zero (