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Cover Page The handle holds various files of this Leiden University dissertation Author: Kalkman, Vincent J. Title: Studies on phylogeny and biogeography of damselflies
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Cover Page The handle holds various files of this Leiden University dissertation Author: Kalkman, Vincent J. Title: Studies on phylogeny and biogeography of damselflies (Odonata) with emphasis on the Argiolestidae Issue Date: 5. redefining the damselfly families: a comprehensive molecular phylogeny of zygoptera (odonata) Klaas-Douwe B. Dijkstra, Vincent J. Kalkman, Rory A. Dow, Frank Stokvis and Jan van Tol Citation: Dijkstra, K.-D.B., V.J. Kalkman, R.A. Dow, F.R. Stokvis & J. van Tol Redefining the damselfly families: the first comprehensive molecular phylogeny of Zygoptera (Odonata). Systematic Entomology, doi: /syen An extensive molecular phylogenetic reconstruction of the suborder Zygoptera of the Odonata is presented, based on mitochondrial (16S, COI) and nuclear (28S) data of 59% of the 310 genera recognized and all (suspected) families except the monotypic Hemiphlebiidae. A partial reclassification is proposed, incorporating morphological characters. Many traditional families are recovered as monophyletic, but reorganization of the superfamily Coenagrionoidea into three families is proposed: Isostictidae, Platycnemididae and Coenagrionidae. Archboldargia Lieftinck, Hylaeargia Lieftinck, Palaiargia Förster, Papuargia Lieftinck and Onychargia Selys are transferred from Coenagrionidae to Platycnemididae, and Leptocnemis Selys, Oreocnemis Pinhey and Thaumatagrion Lieftinck from Platycnemididae to Coenagrionidae. Each geographically well-defined clade of Platycnemididae is recognized as a subfamily, and thus Disparoneurinae (i.e. Old World Protoneuridae ) is incorporated, Calicnemiinae is restricted, and Allocnemidinae (type genus: Allocnemis Selys) subfam.n., Idiocnemidinae (type genus: Idiocnemis Selys) subfam.n. and Onychargiinae (type genus: Onychargia Selys) subfam.n. and Coperini trib.n. (type genus: Copera Kirby) are described. Half of Coenagrionidae belongs to a well-supported clade incorporating Coenagrion Kirby and the potential subfamilies Agriocnemidinae, Ischnurinae and Pseudagrioninae. The remainder is less well defined, but includes the Pseudostigmatidae and New World Protoneuridae that, with Argiinae and Teinobasinae, may prove valid subfamilies with further evidence. Ninety-two per cent of the genera formerly included in the polyphyletic Amphipterygidae and Megapodagrionidae were studied. Pentaphlebiidae, Rimanellidae and Devadattidae fam.n. (type genus: Devadatta Kirby) are separated from Amphipterygidae, and Argiolestidae, Heteragrionidae, Hypolestidae, Philogeniidae, Philosinidae and Thaumatoneuridae from Megapodagrionidae. Eight further groups formerly placed in the latter are identified, but are retained as incertae sedis; the validity of Lestoideidae, Philogangidae and Pseudolestidae is confirmed. For some families (e.g. Calopterygidae, Chlorocyphidae) a further subdivision is possible; Protostictinae subfam.n. (type genus: Protosticta Selys) is introduced in Platystictidae. Numerous new combinations are proposed in the Supporting Information. Many long-established families lack strong morphological apomorphies. In particular, venation is incongruent with molecular results, stressing the need to review fossil Odonata taxonomy: once defined by the reduction of the anal vein, Protoneuridae dissolves completely into six clades from five families. redefining the damselfly families: a comprehensive molecular phylogeny of zygoptera 103 introduction Odonata are among the most ancient of winged insects, dating from the Permian (Grimaldi & Engel 2005). Extant Odonata are considered monophyletic (e.g. Davis et al. 2011) and include two suborders of almost 3000 species each, the Zygoptera or damselflies and the Anisoptera or true dragonflies, and a third suborder, the Anisozygoptera or damseldragons with only four species (Kalkman et al. 2008, Dijkstra et al. 2013). Although wing venation guided classification of Odonata, rampant homoplasy (convergence) obscures relationships, as has been demonstrated in Anisoptera (e.g. Dijkstra & Vick 2006, Ware et al. 2007, Pilgrim & von Dohlen 2008, Fleck et al. 2008a, Blanke et al. 2013). The same applies, perhaps more so, in Zygoptera (O Grady & May 2003, Carle et al. 2008, Pessacq 2008), in which systematic challenges remain in groups with the most simplified venation (mostly Coenagrionoidea) and those characterized by the potentially highly homoplasious insertion of supplementary longitudinal veins (mostly Megapodagrionidae). Although the phylogeny of the Anisoptera has been reasonably well studied and its classification is fairly settled (e.g. Ware et al. 2007, Fleck et al. 2008b), recent studies of Zygoptera rely on rather incomplete molecular datasets (Bybee et al. 2008, Carle et al. 2008, Dumont et al. 2010) and one extensive morphological study (Rehn 2003), although the family Calopterygidae has been studied in detail (Dumont et al. 2005, 2007). Our taxon sampling is the most extensive thus far in Zygoptera, including members of 59% of the 310 genera currently recognized and all (suspected) families, except for the monotypic Hemiphlebiidae. To optimize sampling breadth versus phylogenetic depth, our approach targeted two variable mitochondrial markers [16S, cytochrome c oxidase I (COI)] and a more conserved nuclear one (28S) for many species, rather than more markers for a limited selection. These are among the most commonly applied markers in Odonata and generally provide well resolved and supported trees, at least from species to family level (Hasegawa & Kasuya 2006, Ballare & Ware 2011). Moreover, a relatively long section of 28S was sequenced and the combined total extent of 28S+16S is comparable (84-145%) to several studies with three or more markers (Ware et al. 2007, Bybee et al. 2008, Pilgrim & von Dohlen 2008, Fleck et al. 2008a, b) and 75% of two studies using four nuclear markers only (Dumont et al. 2005, 2010). For 83% of the studied taxa, COI was sequenced and available sequences surpass previous studies ( % and 108%). We focus on the phylogenetic and taxonomic implications of the newly available data (cf. Dijkstra & Kalkman 2012), particularly for the definition of the families, using Silsby (2001) as the basis of the traditional classification (Dijkstra et al. 2013). methods Specimen acquisition The study relies on collections assembled in recent years at the Naturalis Biodiversity Center, Leiden, the Netherlands (formerly the National Museum of Natural History and the Netherlands Centre for Biodiversity Naturalis), by the authors, supplemented with donations from our international network (see the Acknowledgements section). Specimens included in the analysis were collected from 43 countries and from all continents, excluding Antarctica. In 34% of cases, one or two legs were removed from a live sample and preserved in 96% ethanol; the specimen was retained as an acetone-dried voucher. In the remaining cases, legs were removed from a specimen previously preserved either in 96% ethanol or by drying with acetone. dna extraction and amplification Genomic dna was extracted from legs using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Venlo, The Netherlands). Elution was performed in 100 μl elution buffer. Fragments of the nuclear 28S rrna gene ( bp) and the mitochondrial 16S rrna ( bp) and COI genes 104 kalkman studies on phylogeny and biogeography of damselflies (odonata) (658 bp) were amplified using primer combinations developed with Primer3 (Rozen & Skaletsky 2000) (Table S1). Twenty-five microlitres of pcr reaction mixes for 16S and COI contained 2.5 μl of 10 CoralLoad pcr Buffer (Qiagen), 1 μl of each primer (10 pm), 1.25U of Taq dna Polymerase (Qiagen), 0.5 μl of dntps and 1 μl of dna template. Five microlitres of Qsolution (Qiagen) were added to the reaction mix for 28S. The amplification protocol consisted of 3 min at 94 C followed by cycles of 15 s at 94 C, 30 s at 60 C to 35 C and 40 s at 72 C, and a final 5min at 72 C. Direct sequencing was performed at Macrogen Europe on an abi 3730xl sequencer (Applied Biosystems, Carlsbad, ca). Data analysis Sequences were edited with sequencher (Gene Codes Corporation, Ann Arbor, mi) and assembled using bioedit (Hall 1999), geneious pro (Biomatters Ltd, Auckland, New Zealand) (Drummond et al. 2011) was used to check for stop codons. All sequence data and additional geographic and ecological data as well as photographs of the specimens were uploaded to the Barcode of Life Data System (bold; Ratnasingham & Hebert 2007). Sequences were also deposited in GenBank. Barcode index numbers (BINs) and GenBank accession numbers are provided in Table S2. The number of unique site patterns was 635 for 28S, 452 for 16S and 359 for COI. Phylogenetic analyses Multiple sequence alignments were performed using mafft (Katoh et al. 2009) under default parameters. After exploration of all molecular data with neighbour joining analysis using mafft, a selection was made for in-depth analysis of specimens for which both 16S and 28S sequences were available. As a rule, we included two individuals per genus, preferably representing distant species, or two for each distinct clade if the genus appeared not to be monophyletic. The subset included 356 specimens, representing at least 322 species placed in 184 genera. For 295 of these specimens, COI sequences are available as well. Maximum parsimo- ny (mp), maximum likelihood (ml) and Bayesian inference (bi) analyses were performed on the individual datasets of 28S (additional taxa: Amazoneura, Dolonagrion, Megapodagrion) and 16S (adding Coeliccia dinoceras Laidlaw, Xiphiagrion), as well as the combined 28S+16S and 28S+16S+ COI datasets. MP analyses were performed in tnt (Goloboff et al. 2008) by heuristic search with random-taxonaddition replicates, tbr branch swapping, maxtrees set to 1000 with autoincrease. All characters were treated as equal and unordered. Gaps were treated as missing data. Node support was established with a bootstrap analysis of 500 replicates. ML analyses were run with raxml (Stamatakis et al. 2008) using a Gamma model of rate heterogeneity, with each fragment treated as a separate partition. For the bi, the bestfitting nucleotide substitution model for each of the individual fragments was assessed using hierarchical likelihood ratio tests in mrmodeltest 2.3 (Nylander 2004). For 28S and 16S, a general time reversal (GTR + I + G) model (nst=6) was selected, whereas for COI the Hasegawa-Kishino-Yano model (nst=2) was used, all with a proportion of invariable sites and a gamma distribution for rates across sites (rates = invgamma). For each dataset, two independent Monte Carlo Markov Chain simulations were run in mrbayes (Huelsenbeck & Ronquist 2001) with four chains, for generations and a sample frequency of 2000 at a temperature of 0.05, providing trees for the consensus after an average standard deviation of split frequencies 0.01 had been reached. Several anisopterans were tested as outgroup, leading to similar topologies, and thus only results using Aeshna juncea (Linnaeus) of Aeshnidae are shown. Morphology Specimens in the Naturalis Biodiversity Center (Leiden) and University Museum of Zoology (Cambridge) and numerous publications (e.g. Bechly 1996, O Grady & May 2003, Rehn 2003, Gassmann 2005, Bybee et al. 2008, Pessacq 2008, van Tol et al. 2009, Garrison et al. 2010, Yu & Bu 2011b) were examined for morphological redefining the damselfly families: a comprehensive molecular phylogeny of zygoptera 105 characters of the lineages identified by molecular analysis. results The phylogenies reconstructed on the 28S+16S and 28S+ 16S+COI datasets are presented in figures 1-3. Support for important clades is summarized in Table S3 and, where relevant, is discussed for separate partitions in the following sections. Of the generally accepted families, many were recovered as monophyletic with good support in (almost) all analyses: Calopterygidae, Chlorocyphidae, Euphaeidae, Isostictidae, Lestidae, Lestoideidae, Platystictidae and Polythoridae. Coenagrionidae was monophyletic if the Pseudostigmatidae and New World Protoneuridae were included, as was Platycnemididae if the Old World Protoneuridae were included, although seven genera had to be moved between the two families (see Discussion for details). Amphipterygidae and Megapodagrionidae were found to be highly polyphyletic, although the latter revealed several large and sometimes wellsupported groups of genera. The remaining families are either monogeneric (Philogangidae, Pseudolestidae) or nearly so with only one taxon sampled (Dicteriadidae). Perilestidae and Synlestidae were paraphyletic in many analyses but in BI/ML 28S+16S+COI, the two combined were monophyletic with low support and a well-supported monophyletic Perilestidae was embedded in Synlestidae. Although family-level clades often were well supported and the internal topology of these clades was frequently resolved, generally limited support was found for family interrelationships. However, the clade combining Coenagrionidae, Platycnemididae, Pseudostigmatidae and the entire Protoneuridae was well supported. BI/ML 28S+16S recovered Isostictidae as their sister group, but with low support. Lestoidea (including Lestidae, Perilestidae and Synlestidae) was well supported consistently as the sister group of all remaining Zygo ptera, as was Platystictidae as the sister group of the remainder. No nearest relatives could be identified for the families Calopterygidae, Chlorocyphidae, Philogangidae, Polythoridae and Pseudo lestidae, as well as for the clades that formerly constituted Amphipterygidae and Megapodagrio nidae. Only Euphaeidae and Lestoideidae appeared consistently as sister groups with good support, but their further relationships remained unclear. The results and their implications are detailed in the following section for each family and/or well-supported clade. discussion Molecular and morphological studies indicate that Zygoptera are monophyletic (Bechly 1996, Rehn 2003, Bybee et al. 2008, Carle et al. 2008, Dumont et al. 2010), although Trueman (1996, 2007) questioned this based on an analysis of wing venation. Given our focus on that suborder and the often low support of higher-level relationships found within it, our dataset is unsuited for this issue. Our analyses supported the prevailing family classification for 72% of all genera and 80% of all species of Zygoptera. Therefore recognition at the family level of equally well-supported but previously unrecognized clades, particularly within the family Megapodagrionidae, should be considered. Proposed taxonomic consequences are discussed in the following sections, starting with the relatively well defined smaller sister groups of remaining Zygoptera (the superfamilies Lestoidea and Platystictoidea), followed by the crown radiation of the Coenagrionoidea, which encompasses three-fifths of all damselfly species. The discussion concludes with the problematic remainder, grouped strictly for convenience in the probably paraphyletic Calopterygoidea. The suggested reclassification of Zygoptera based on this discussion is provided in Appendix 1, including the authorities for all genus- and familygroup names. Diagnoses of new or revised family groups are given in Appendix 2 and a summary of proposed taxonomic changes, including new combinations, is given in Table S kalkman studies on phylogeny and biogeography of damselflies (odonata) Figure 1. Summary of Zygoptera phylogeny, based on figures 2, 3 and the Discussion section. Only reasonably supported dichotomies are shown. The classification follows Appendix 1 (see for other genera placed near Dimeragrion, Priscagrion and Rhipidolestes) and the fate of some traditional taxa is indicated. For each recognized damselfly lineage, the known numbers of genera and species (in brackets) are shown, as is their occurrence in the Afrotropical (at), Australasian (au), Nearctic (na), Neotropical (nt), Oriental (ol), Pacific (pc) and Palaearctic (pa) regions. redefining the damselfly families: a comprehensive molecular phylogeny of zygoptera 107 Figure 2a. Phylogenetic reconstruction for 295 specimens from the combined Bayesian analysis of 28S, 16S and COI. Posterior probabilities are shown (as percentages) only if below 100%. Species names and classification as proposed are shown. (a) Lestoidea and Platystictoidea; (b, c) various groups; (d) Platycnemididae; (e) Coenagrionidae. Superfamily Lestoidea All analyses confirmed that the over 200 species of Lestoidea (not to be confused with the unrelated genus Lestoidea) form the sister group of all the other 93% of damselflies (cf. Bybee et al. 2008, Carle et al. 2008, Dumont et al. 2010, Davis et al. 2011). Contrary to Rehn (2003), the position of the superfamily Lestoidea suggests that the narrowed external edge of the labial palp is an apomophy of remaining Zygoptera, rather than that the expanded edge arose convergently in Lestoidea and Anisoptera. With the exception of Hemiphlebia, Lestoidea possesses distinctly modified secondary genitalia with a reduced apex of the genital ligula (the functional penis) and triangular anterior hamules (Rehn 2003, Garrison et al. 2010). The monotypic Hemiphlebiidae from southeastern Australia and Tasmania was not studied, but is the sister group of remaining Lestoidea according to previous studies (Rehn 2003, Dumont et al. 2010, Davis et al. 2011). Just over a quarter of the Lestoidea species are placed currently in the Perilestidae and Synlestidae. 108 kalkman studies on phylogeny and biogeography of damselflies (odonata) Figure 2b. Continued. We found no support for the monophyly of the latter family, but in BI/ML 28S+16S+COI the two combined were monophyletic with low support, as was Perilestidae with high support. The last is expected given the morphological similarity of the Neotropical Perilestes and Perissolestes. The monotypic Nubiolestes from central Africa has been considered the only non-american perilestid, but formed the sister group of the American genera only in BI/ML 28S+16S+COI, although with low support. However, we had difficulty amplifying COI for many synlestids and BI, ML and MP of 28S and 28S+16S recovered Nubiolestes as the sister group of the southern African Chlorolestes. Neotropical Perilestidae share mid-dorsal spines on the larval abdomen, a two-toothed ovipositor, redefining the damselfly families: a comprehensive molecular phylogeny of zygoptera 109 Figure 2c. Continued. very short pterostigmata, anal veins reduced basally (or shifted distally) allowing the quadrilateral cells to reach the wing margins, and a distinctive layout of markings and appendages (Garrison et al. 2010). The anal vein and ovipositor dentition of Nubiolestes are intermediate to Chlorolestes and other features are closer to the latter. Interestingly, the Synlestidae from South Africa (Chlorolestes, Ecchlorolestes) never grouped together, nor did those from Australia (Episynlestes, Synlestes). We did not sample the problematic genera Phylolestes from Hispaniola, Sinolestes from China and Chorismagrion from Australia. The last, sometimes placed in the Chorismagrionidae (Bechly 1996), was recovered within Synlestidae by Bybee et al. (2008) and Dumont et al. (2010). May et al. (unpublished data in Dijkstra et al. 2013) found Synlestidae to be monophyletic if Nubiolestes was included, but morphological apomorphies for the group remain to be identified. Given the discussed problems, we retain Perilestidae and Synlestidae as currently recognized, only transferring Nubiolestes, although Synlestidae may still prove not be monophyletic and the two families might eventually be merged or divided. Lestidae was monophyletic in all analyses (cf. Rehn 2003, Bybee et al. 2008, Dumont et al. 2010). Austrolestes and Indolestes are sister groups, but were never monophyletic with Sympecma, which also closes its wings at rest, and thus no support for the subfamily Sympecmatinae was fo
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