Life on the : Ecology and Evolution of the Helicopter

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Spencer J. Ingley [email protected] (352) 278-2705 Department of Wildlife Ecology and Conservation Advisor: Dr. Marc Branham CALS Honors Program

ABSTRACT

Helicopter damselflies (Pseudostigmatidae: Odonata) form a relatively small, yet dynamic group of endangered odonates (including the largest extant odonate, Megaloprepus caerulatus, with a wingspan of ~190 mm). This highly specialized group is found in primary- growth rainforest (Central and South America; one East African ) where they oviposit exclusively in phytotelmata and are specialist foragers on orb weaver which are plucked from their web. Pseudostigmatids exhibit unique wing structure within Zygoptera, and within

Pseudostigmatidae both broad and narrow wing forms exist. Oviposition, feeding, and wing form evolution are examined for the first time within an evolutionary context using modern phylogenetic methods of reconstruction and character optimization. Phylogenetic analyses

(Bayesian and Parsimony) were performed on a data set composed of 60 morphological characters and ~3.4kb of sequence data (Mitochondrial loci: 12S, 16S, COII; Nuclear loci: 28S,

H3). Findings include monophyletic Pseudostigmatidae, (East African species) as the sister group to all Neotropical genera, and Pericnemis as sister to

Pseudostigmatidae. The genera and are monophyletic while

Microstigma forms a monophyletic group with Megaloprepus. Oviposition in phytotelmata likely evolved multiple times within Zygoptera, and spider feeding evolved from a single origin. There are two separate origins of narrow wings within Pseudostigmatidae. These findings provide new insight into Pseudostigmatid evolution that can be used to generate awareness of the threatened status of Helicopter damselflies.

KEY WORDS

Helicopter Damselflies, Odonata, Behavioral evolution, wing morphology, oviposition, spider feeding. INTRODUCTION

Odonata, the order that is comprised of Damselflies (Zygoptera) and

(), is one of the most ancient winged insect groups (~300 MYA). Odonata is comprised of roughly 5,500 species, and has a worldwide distribution. Odonates are among the most acrobatic flyers in the world. Using four wings capable of moving independently, they are equally adept at snatching either a mate or prey out of the air. Of the known species, over 600 are listed on the International Union for Conservation of Nature (IUCN) Red List for endangered and threatened species (IUCN Red List Website, http://www.iucnredlist.org/search). For these and other reasons, odonates are gaining momentum as an organism important to conservation efforts worldwide (Polhemus, 1997;

Samways and Steytler, 1996; Painter,

1999; Hawking and New, 1999). Perhaps the biggest impediment to their success as a conservation tool is the lack of knowledge regarding the classification and evolution of taxa below the family level.

For effective conservation of biodiversity Ingley 2009 to take place, it is essential that species are Fig.1. (a) Megaloprepus caerulatus: the largest extant Odonate species, which belongs to the family properly identified and classified. Among Pseudostigmatidae; (b) Teinobasis aerial: a „typical‟ in terms of size and morphology. the most spectacular of all odonates are the

Helicopter damselflies (Zygoptera: Pseudostigmatidae; Figure 1a.). This group is composed of 6 genera and 19 species. Nearly all species of pseudostigmatids inhabit Neotropical rainforests, with one species endemic to east Africa (Coryphagrion grandis). The group is famous for its gigantism and includes the largest extant odonate, Megaloprepus caerulatus, with a wingspan of over 190mm (Groeneveld et al., 2007; Figure 1a.). The evolution of damselfly gigantism has been examined phylogenetically and it is hypothesized to have a single origin attributed only to

Helicopter damselflies (Groeneveld et al., 2007).

Oviposition Behavior

In addition to their extreme size, pseudostigmatids exhibit unique oviposition and feeding behaviors. While most odonates oviposit endophytically in lake and stream vegetation, pseudostigmatids oviposit exclusively in phytotelmata (rain filled, naturally occurring water containers including tree-holes, , tank bromeliads, and fruit husks; Figure 2) (Corbet,

2004). Though female pseudostigmatids typically land inside or along the margins of

Collins 2009

Fig. 2. Megaloprepus caerulatus ovipositing in a tree hole in Costa Rica. This species, along with other members of Pseudostigmatidae, exhibit this behavior of phytotelmata oviposition. Other oviposition habitats include tank bromeliad plants, bamboo internodes, and fruit husks. phytotelmata and deposit their eggs in the water collected within the container, some species have been observed “launching” their eggs into phytotelmata while hovering above (Machado and Martinez, 1982). These behaviors, as well as their unique larval habitat, have been studied extensively for a small number of species (Fincke, 2005; Machado and Martinez, 1982) and documented for nearly all (Fincke, 2005; Corbet, 2004), but have never been examined in an evolutionary context using modern phylogenetic methods.

Feeding Behavior

While adult odonates typically feed on small flying captured while in flight, pseudostigmatids (the only unequivocal specialist foragers within Odonata; Corbet, 2004)

Collins 2009

Fig. 3. approaching an orb-weaver spider web. This species, along with other Pseudostigmatid species, are the only known unequivocal specialist foragers within Odonata, feeding exclusively on orb-weaver spiders plucked from their web. specialize on orb-weaver spiders, which they pluck directly from their webs (Figure 3).

Pseudostigmatids have been observed plucking insects caught in webs as well (Corbet, 2004). In

addition, pseudostigmatids are known to defend a set of webs within an established territory, a

behavior that resembles a sort of harvest tactic, as orb-weaver spiders will use a vacant web

instead of spinning a new one (Corbet, 2004). The behavior of web feeding, both on spiders and

Collins 2009 their prey caught in the webs, have yet to be examined using modern phylogenetic methods.

Wing Form

In most zygopterans, including Pseudostigmatidae, a narrow petiolate wing form is common

(Figure 4a). However, four pseudostigmatid genera (, , Megaloprepus,

and Pseudostigma) each exhibit an expanded or broadened wing beyond the basal petiolation

(Figure 4b). This broadening of the wing is specifically defined as having expansions of

intercalated veins and/or dichotomous branching of radial veins from the RP2 vein forward

through the CuA vein (Figure 4a, 4b). The extent of the expansion varies among genera. Other

groups within Zygoptera exhibit similar wing expansions (i.e. Calopterygiodea) but the origins

and directionality (as well as the functional role) of this trait have never been examined.

Due to their high level of behavioral and wing structure specialization and their importance

to conservation (4 of 19 species of pseudostigmatids are thought to be threatened or endangered;

IUCN Red List Website, http://www.iucnredlist.org/search) study of the group is especially

urgent. Here, we present the first comprehensive analysis of pseudostigmatid phylogeny, and

use this phylogeny to examine the origins and evolution of these unique behavioral and

morphological adaptations.

Ingley 2009

a.

Ingley 2009

b.

Fig. 5. Wing forms present in Pseudostigmatidae, showing characterization of the expanded wing form. This expansion or broadening of the wing is herein defined as having expansions of intercalated veins and/or dichotomous branching of radial veins from the RP2 vein forward through the CuA vein. (a) Narrow wing of Mecistogaster lucretia, lacking major innovation of pterostigma and wing expansion. (b) Broad wing for of Microstigma anomalum, showing modified pterostigma and expanded wing form.

MATERIALS AND METHODS

Taxon sampling

Of the 19 recognized species that comprise Pseudostigmatidae, 16 were used in this analysis (Appendix I). Several species in the family, largely due to their conservation status or difficulty in attaining permits in the country of origin (e.g., Brazil), were not available to generate either morphological or molecular data. However, the 16 species included herein represent all six currently recognized pseudostigmatid genera, both from the Old World and New

World. Multiple exemplars were obtained and used for morphological character coding (except for Coryphagrion grandis, which I coded from the literature and photographs taken by Seth

Bybee of specimens in the collection of The Natural History Museum, London). Outgroups were selected from ten closely related genera, resulting in 14 outgroup taxa (Appendix I). The outgroup sampling was focused on including other coenagrionoid zygopteran taxa that inhabit similar niches (e.g., living in the forest understory) and those exhibiting similar behaviors as pseudostigmatids (i.e., use of phytotelmata as larval habitat). Outgroup taxa represented species from Southeast Asia, the South Pacific, the Caribbean and North, Central, and South America.

Lestes disjunctus served as the root taxon for all analyses due to knowledge gained from prior analyses, which provide significant evidence supporting as sister to all other zygopterans (Bybee et al., 2008; Carle et al., 2008). This extensive outgroup sampling was selected due to the uncertainty surrounding the sister group of Pseudostigmatidae, as well as its placement within Zygoptera and to better explore the evolutionary history of the group‟s unique behaviors.

Laboratory methods

Genomic DNA was extracted from both dry museum specimens and ethanol preserved specimens, using Qiagen Dneasy protocol for tissues (Valencia, CA). I extracted muscle tissue from the thoracic region and/or a leg. Specimens used for DNA extraction were provided by the Florida State Collection of (Gainesville, FL) and the Insect Genomics

Collection (IGC), M.L. Bean Museum, Brigham Young University. All specimens from the IGC are preserved in 95% ETOH and stored at -80°C.

The molecular data set used for this analysis was comprised of five genes: 12S ribosomal

(12s rDNA, 0.4 kilobase pairs (kb)), 16S ribosomal (16S rDNA, 0.6kb) and the protein coding gene cytochrome oxidase subunit II (COII, 0.6kb) from the mitochondrion, and 28S ribosomal

(28S rDNA, 1.3kb), and Histone 3 (H3, O.5 kb) from the nucleus. Primers are given in Whiting

(2001), Bybee et al. (2004), Ogden and Whiting (2005), and Terry and Whiting (2005). The 28S rDNA, COII and H3 genes were each amplified using a three-step PCR at 40 cycles with an annealing temperature of 54 °C for 28S rDNA and 50 °C for COII. The 12S rDNA and 16S rDNA gene were amplified using a touchdown method with the annealing temperature starting at

62 °C and decreasing to 42°C over 40 cycles of a standard three-step PCR. All PCR products were visualized via agarose gel electrophoresis to assure proper amplification and detect possible contamination using negative controls. Products were purified using a Montage PCR Cleanup

Kit (Millipore) and cycle-sequenced using BigDye Terminator chemistry (ABI). Sequences were generated using an ABI 3100 capillary sequencer at the DNA Sequencing Center, Brigham

Young University. Complementary strands were sequenced with sufficient fragment overlap to reduce sequencing errors. The nucleotide-nucleotide BLAST (blastn) search function on

GenBank (http://www.ncbi.nlm.nih.gov/) was used to examine all sequences for contamination.

In addition to molecular sequence data generated, 23 morphological characters from Rehn

(2003), combined with 37 original characters that I derived were coded for each taxon to provide more phylogenetic resolution for this species level analysis. Characters were derived from wing venation, skeletal, and behavioral attributes totaling 60 morphological characters (Appendix II).

Data Analysis

All phylogenetic topologies presented in this paper result from a combination of both morphological and molecular data, forming a total evidence analysis (TEA) of both data sets.

TEA provides the opportunity for both molecular and morphological data partitions to inform each other such that hidden underlying signal among all data sets is discovered (Wheeler et al.,

2001; Ogden and Whiting, 2003; Pilgrim et al., 2003; and Bybee et al., 2008). The alignments for COII and H3 were generated in Sequencher 4.1 (GeneCodes, 2002, Sequencher v4.5, Ann

Arbor, MI) based on conservation of codon reading frame. Sequences for the ribosomal genes were initially aligned manually in Sequencher 4.1 to identify conserved and variable regions. These regions were then subdivided into partitions in order to assist the search strategy in finding more optimal solutions in MUSCLE 3.7 (Edgar, 2004)

Analyses were performed using both Bayesian as Parsimony methods. Bayesian analyses were run in MrBayes 3.1.1 (Ronquist and Huelsenbeck 2003). Using Modeltest (Posada and

Crandall 1998), the GTR+I+G was selected as the best-justified model to represent the molecular data and the Mk model was used to model the morphological data. The first 20,000 were discarded as “burn-in” as determined by graphing the generations in Tracer 1.4.1 (Rambaut and

Drummond, 2007). The data matrix was analyzed for 20,000,000 generations to achieve the current topology. Partition Bremer support values were computed via PAUP*.4.0b10 using a command file generated in TreeRot 2Vc (Sorenson, 1999). Bootstrap values were also generated via PAUP*.4.0b10. Parsimony analyses were performed via TNT (Goloboff et al., 2003) using a new technologies search using a ratchet, TBR, and SPR tree searching strategies.

Characters related to spider-feeding, oviposition in phytotelmata, and the presence of the expanded wing condition were included within the TEA and optimized using the default parameter settings in MacClade 4.06 OS X. All characters were explored with alternative optimizations in MacClade (ACCTRAN or DELTRAN). Characters related to spider-feeding and oviposition in phytotelmata, as well as those related to the expanded wing condition can be found in Appendix II.

RESULTS

Parsimony analysis

In the TEA two most parsimonious trees were recovered (CI= 0.38; RI=0.73; Figure 5).

Pseudostigmatidae was supported as monophyletic, with the East African Coryphagrion grandis as sister to all Neotropical genera. The Pericnemis was recovered as sister to the ingroup, all genera except Microstigma were supported as monophyletic, with Megaloprepus breaking up

Microstigma.

Fig. 5. Strict consensus of the two most-Parsimonious trees resulting from the total evidence analysis of Pseudostigmatidae + outgroups. The tree was rooted to disjunctus. Branch support values are given above and below nodes. Boot Strap support values are shown above the nodes, and Bremer support values are shown below. CI: 0.38; RI: 0.73.

Bayesian analysis

Bayesian analyses were also performed on the combined data set using the Mk model for morphology (Ronquist et al., 2005) and the GTR+I+G model for the molecular data set (Figure

6). Relationships within Pseudostigmatidae were similar to the parsimony topology. Differences included Microstigma as sister to the clade formed by Megaloprepus + Anomisma. The genus

Mecistogaster was not recovered as monophyletic, due to Pseudostigma nesting within the clade comprised of Pseudostigma +Mecistogaster. Compared with the Parsimony analysis, the

Bayesian analysis recovered less resolution among outgroup taxa, and no clear sister group to a monophyletic Pseudostigmatidae was recovered.

Fig. 6. Bayesian phylogeny resulting from total evidence analysis of Pseudostigmatidae + outgroups, rooted to Lestes disjunctus. Boot Straps branch supports are above nodes. DISCUSSION

Monophyly and Sister Group of Pseudostigmatidae

This study supports the monophyly of Pseudostigmatidae. The relationship between

Paleotropical and Neotropical genera was strongly supported in both Bayesian and Parsimony analyses, providing stronger evidence that Coryphagrion is a Gondwana relict, and indeed a member of Pseudostigmatidae. For some time it was thought that Coryphagrion had evolved convergently, and that the ecological and morphological similarities that are shared with

Neotropical Pseudostigmatids were the result of a similar habitat, and not a shared common ancestry. However, my analysis provides additional support for the hypothesis that

Coryphagrion shared a recent common ancestor with Neotropical Pseudostigmatids before

Gondwana split to form the American and African continent sometime in the early Cretaceous

(~130 MYA). The isolated East African distribution of Coryphagrion is likely much smaller than its historic distribution, which likely extended throughout Africa towards the Western

Coasts in what were then contiguous forests. Coryphagrion was also supported as sister to

Neotropical genera in Groeneveld et al. 2007, though a smaller data set than that of this study was used (two mitochondrial markers, 16S and ND1, and one nuclear marker, EF1).

There has been some debate regarding the placement of Pseudostigmatidae within

Zygoptera (Bybee et al. 2008). Though phylogenetic relationships in the Bayesian analysis were highly resolved within Pseudostigmatidae, it failed to recover any highly supported sister group.

However, the Parsimony analysis recovered the genus Pericnemis as sister (P. strictiva & P. incallida; as sister to Pseudostigmatidae).

Relationships among Neotropical Pseudostigmatidae

Two major Neotropical clades were supported in both parsimony and Baysian analyses:

Anomisma + Megaloprepus + Microstigma, and sister clade Pseudostigma + Mecistogaster.

Megaloprepus caerulatus formed a polytomy with the genus Microstigma in the Parsimony analysis, while it was recovered as sister to Anomisma in the Bayesian analysis, with the genus

Microstigma fully resolved and sister to Anomisma + Megaloprepus. Both Pseudostigma and

Mecistogaster were supported as monophyletic in the Parsimony analysis, while in the Bayesian analysis Pseudostigma was nested in Mecistogaster. The findings from my Parsimony analysis support those reported in the combined Maximum Parsimony analysis of Groeneveld et al. 2007, for which a total of five species from the genera Coryphagrion, Megaloprepus, Pseudostigma, and Mecistogaster were used to infer the evolution of gigantism.

Behavioral Evolution: Oviposition in Phytotelmata and Spider-feeding

It was speculated by Calvert (1911) that the use of phytotelmata as larval habitat for odonates originated in flooded forests of the Amazon, where at high water, low hanging bromeliads might have been mistaken for aquatic vegetation. The use of tree holes might have originated from similar oviposition „mistakes‟ in the holes of trees that fall over or trees near streams or other bodies of water (Fincke, 2006). I observed multiple individuals from the genus

Microstigma, both male and female at multiple localities in Ecuador (Limoncocha, Rio Bigal), frequenting felled trees cut by humans. These trees were not located near any water source, so presumably these individuals cued in on the large gap in the forest created by the felled trees and were looking for phytotelmata habitat (personal observation). In addition, I observed two females of distinctive Mecistogaster species flying among patches of bamboo, presumably looking for suitable sites to oviposit (personal observation). Though the specific selection pressures that may have influenced the evolution of this behavior are uncertain, my studies

support the hypothesis that oviposition in phytotelmata has a single origin. Based on my current

taxon sampling, I propose that oviposition in phytotelmata by pseudostigmatids and other closely

related zygopterans is a result of close genealogical relationship, and not convergent evolution

(Figure 7). Though my sampling does not include all zygopterans that oviposit in phytotelmata

(see next paragraph), my data clearly support a single origin of this unique behavior within the

clade that includes Pseudostigmatidae. However, this behavior seems to have been lost

subsequently in at least two separate lineages (Teinobasis and Pericnemis, Figure 7), perhaps due

to selection pressures influenced by changing ecosystem structure or intense competition for the

limited amount of phytotelmata habitat available.

Fig. 7. Strict consensus of the two most-Parsimonious trees resulting from the total evidence analysis of Pseudostigmatidae + outgroups. The tree was rooted to Lestes disjunctus. Black branches represent taxa that oviposit endophytically in streams, rivers, or ponds. Blue branches represent taxa that oviposit in phytotelmata. Black arrows indicate a proposed origin in this behavior, with one basal origin, and two subsequent origins/losses. The blue arrow represents the single origin of phytotelmata oviposition behavior. Other possible hypotheses for the evolution of oviposition in phytotelmata could also

prove valid. For example, this behavior likely evolved separately in members of the genus

Megalagrion. Though the species from this genus that was included in my analysis

(Megalagrion blackburni) does not exhibit this behavior, other members of this genus, M.

koelense and M. kuaiense, do oviposit in phytotelmata (Polhemus and Asquith, 1996; Polhemus,

1997). This genus has been supported as monophyletic in previous studies (Polhemus, 1997).

This suggests that oviposition in phytotelmata has evolved and/or been lost multiple times, likely

due to similar habitats and morphology. Further studies with more expansive taxon samplings

are needed in order to test competing hypotheses.

Spider-feeding, defined as the plucking of spider prey from their webs, evolved once in

Zygoptera (Figure 8). My results support a single origin of this unique feeding behavior, known

Fig. 8. Strict consensus of the two most-Parsimonious trees resulting from the total evidence analysis of Pseudostigmatidae + outgroups. The tree was rooted to Lestes disjunctus. Black branches represent taxa that do not spider-feed (i.e., pluck spiders from their web). Blue branches represent taxa that exhibit the spider-feeding behavior. The black and blue arrows represent the origin of non-spider-feeding and spider- feeding respectively. Spider-feeding was supported as having one origin, and is unique to Pseudostigmatidae. only to occur in Pseudostigmatidae. However, the selective forces of evolution favoring this behavior are not well understood. Other species are known to eat spiders on occasion, but this behavior involves individuals snatching spiders from branches or leaves, and not from their web.

This behavior is likely a result of opportunistic feeding. Further studies including other species that have been observed eating spiders under different circumstances are needed to tease apart the evolution of this behavior further.

Expanded Wing Form

Based on findings from my analysis, I propose two possible evolutionary scenarios regarding the evolution of the expanded wing form. Both scenarios support the hypothesis that the expanded wing forms seen in the family and that present in

Pseudostigmatidae are a result of convergent evolution, and not close genealogical relationship.

However, the two scenarios proposed in the following sections differ in the evolution of the expanded wing within Pseudostigmatidae. Little is known about the evolutionary forces driving this wing formation. Colleagues and I are currently exploring two possible explanations. One explanation is related to aerodynamic and flight benefits related to the expanded wing and loss of pterostigma. The other possible explanation is associated with the color patterns in the wings, and their role in reproduction.

One possible scenario for the evolution of the expanded wing in Pseudostigmatidae is that of two separate origins within the family: one in the clade comprised of Anomisma

+Microstigma +Megaloprepus, and the other in the genus Pseudostigma (Figure 9a). This scenario also suggests that the narrow wing form exhibited in the genus Mecistogaster is homologous with the narrow wing form exhibited in other, more basal zygopterans and pseudostigmatids. a.

b.

Fig. 9. Phylogenetic representation of one hypothesis of wing form evolution in Pseudostigmatidae. (a) This represents the scenario in which the expanded wing form had two separate origins: one in the clade comprised of Anomisma + Megaloprepus +Microstigma (red branches), and the other in the genus Pseudostigma (blue branches). Black branches represent the basal condition (i.e., narrow wing form), which is exhibited in Coryphagrion and Mecistogaster genera. (b) This represents the scenario in which the expanded wing form one origin and one subsequent loss. Black branches represent the basal narrow wing condition. Red branches represent taxa that have an expanded wing form. Blue branches represent taxa that have secondarily lost the expanded wing form, and returned to the narrow wing form. This scenario suggests a gradient of evolution, with a rapid expansion of the wing followed by a gradual return to the narrow wing form.

The other possible scenario for the evolution of the expanded wing form in

Pseudostigmatidae suggests one origin and one subsequent loss (Figure 9b). I favor this scenario because it shows an evolutionary gradient from the highly expanded wing form found in

Megaloptepus and Microstigma to the less expanded form found in Pseudostigma, and finally a return to the narrow wing form found in Mecistogaster.

CONCLUSIONS

This research represents the most comprehensive phylogeny of Pseudostigmatidae to date and is a crucial step in understanding the evolution and phylogeny of this dynamic group, which is needed for furthering conservation efforts surrounding the group. With global climate change predicted to affect precipitation patterns in the tropics, as well as continuing deforestation, pseudostigmatid habitat is under great threat. Understanding the directionality of the evolution of the behaviors unique to Pseudostigmatidae could contribute to making more accurate predictions regarding their future range and ability to adapt, thus providing needed focus for conservation efforts.

It is possible that as deforestation in the tropics continues, phytotelmata habitat suitable for use by these charismatic insects will slowly diminish, and may eventually disappear altogether, resulting in extinctions and/or a shift towards other larval habitats by pseudostigmatids and related Odonates. For this reason, pseudostigmatids could serve as a charismatic invertebrate poster child for rainforest conservation.

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APPENDIX I

List of all taxa included in this analysis.

Family Species

Outgroups

Calopterygidae aequabilis

Lestidae Lestes disjunctus

Coenagrionidae resolutum

Coenagrionidae Megalagrion blackburni

Coenagrionidae salva

Coenagrionidae Diceratobasis melanogaster

Coenagrionidae Teinobasis ariel

Coenagrionidae Teinobasis fortis

Coenagrionidae Leptagrion macrurum

Coenagrionidae Leptagrion perlongum

Coenagrionidae Leptagrion fernandezi

Coenagrionidae Pericnemis strictica

Coenagrionidae Pericnemis incallida

Protoneuridae Roppaneura beckeri

Ingroups

Pseudostigmatidae Coryphagrion grandis

Pseudostigmatidae Anomisma abnorme

Pseudostigmatidae Megaloprepus caerulatus

Pseudostigmatidae Microstigma maculatum Pseudostigmatidae Microstigma anomalum

Pseudostigmatidae Microstigma rotundatum

Pseudostigmatidae Pseudostigma aberrans

Pseudostigmatidae Pseudostigma accedens

Pseudostigmatidae Mecistogaster ornate

Pseudostigmatidae

Pseudostigmatidae

Pseudostigmatidae Mecistogaster lucretia

Pseudostigmatidae Mecistogaster jocaste

Pseudostigmatidae Mecistogaster astricta

Pseudostigmatidae Mecistogaster buckleyi

Pseudostigmatidae Mecistogaster martinezi

APPENDIX II

Morphological Matrix Characters

1. Shape of clypeus: (0) rectangular, with anteclypeus and postclypeus forming distinct anterior and dorsal faces, respectively; (1) flattened, with anteclypeus tilted back and not distinct from dorsal facing postclypeus; (2) greatly swollen and rounded into prominent snout; (3) vertical, with anteclypeus and postclypeus facing anteriorly.

2. Shape of frons: (0) smoothly rounded in profile; (1) angulated; (2) flattened; (3) grossly enlarged, forming most of the head anterior to the eyes.

3. Ecdysial cleavage line: (0) well developed; (1) partially developed; (2) absent.

4. Postfrontal suture: (0) vestigial or absent; (1) partially developed; (2) well developed.

5. Position of IR1: (0) closer to RP1 than to RP2; (1) equidistant from RP1 and RP2; (2) closer to

RP2 than to RP1.

6. Postnodal crossveins: (0) unaligned in the C–RA and RA–RP spaces; (1) aligned in the C–RA and RA–RP spaces only; (2) aligned in a transverse series to beyond IR2

7. Pterostigma (Pt): (0) absent; (1) present in only the C–RA space; (2) secondarily lost in both sexes and replaced by a densely reticulate network of veins.

8. Stigma brace vein: (0) absent; (1) present.

9. Position of RP midfork: (0) located beyond 25% wing length; (1) located at less than

25% wing length.

10. IR2: (0) apparently joined to RP' with a crossvein; (1) fused directly to RP' at an acute angle, or with a gentle forward curve.

11-15. CuA: (0) not forked throughout its entire length; (1) long and pectinate distally; (2) reduced and ending on the posterior side of the quadrangle.

Character

State 13 14 15 16 17

0 1 0 0 0 0

1 1 0 1 0 0

2 1 0 0 0 1

16. Discoidal cell: (0) Acute; (1) short square; (2) long square; (3) acute in fore wing, obtuse in hind wing; (4) obtuse in both pairs of wings.

17. Position of nodus: (0) located at one-third to one-half wing length; (1) located at one-quarter to one-third wing length; (2) located at less than one-quarter wing length.

18. RP1–IR1field: (0) expanded and filled by intercalated veins; (1) narrow, with no RP1 branches or intercalated veins.

19. IR1–RP2field, expanded intercalated sectors: (0) absent; (1) present.

20. RP2–IR2field, expanded intercalated sectors: (0) absent; (1) present.

21. IR2–RP3field: (0) expanded and filled by intercalated veins; (1) narrow, with no RP3 branches or intercalated veins.

22. RP3–MA field: (0) expanded and filled by intercalated veins; (1) narrow, with no MA branches or intercalated veins.

23. MA–MP field: (0) expanded and filled by intercalated veins; (1) narrow, with no MA branches or intercalated veins.

24. MP–CuA field: (0) expanded and filled by intercalated veins; (1) narrow, with no CuA branches or intercalated veins.

25. Basal proximity of IR2and RP3: (0) not positioned extremely close to one another near their origins for the length of several cells; (1) positioned extremely close to one another basally for the length of several cells.

26. Apices of RA and RP1: (0) meeting the distal wing margin anterior to the apex of the wing itself; (1) meeting the distal wing margin posterior to the wing apex.

27. Paraprocts: (0) simple, unmodified lobes projecting from sternum of segment 10; (1) modified into 'inferior appendages' for grasping females.

28. Length of abdomen: (0) not greatly elongated for oviposition in phytotelmata (tank bromeliads, water filled tree holes, etc.); (1) abdomen extremely elongated (total length at least

62 mm, but usually >80 mm) as a modification for oviposition in phytotelmata.

29. Additional crossveins present immediately distal and/or basal of Cu crossing: (0) absent; (1) present

30. Field between RP1 and RP2 filled with more than two cells: absent (0); present (1)

31. Field between CuA and wing hind margin: (0) less than 2 cells; (1) 2 cells with 3 cells occasionally 3 cells; (2) greater than 2 cells

32. Triadic branching of veins CuA and MA: absent (0); present (1)

33. RP3/4 with apical fork: absent (0); present (1)

34. Large part of post-subnodal space, subdivided into more than 2 cells: absent (0); present (1)

35. CuA, MA and MP long ending near wing apex: absent (0); present (1)

36. Adults inhabit forest understories: absent (0); present (1).

37. Prey on spiders and small insects from spider webs: absent (0); present (1)

38. Strong intraspecific/ interspecific larval competition and cannibalism: absent (0); present (1)

39. Male superior appendages: superior appendages shorter than inferior (0); superior appendages longer than inferior (1); approximately the equal in length

40. Abdominal segment 10, viewed laterally: as wide as it is high (0); higher than wide (1)

41. Inferior appendages: pointed, sometimes small but present (0); blunt, but distinguishable from the side or very small almost rudimentary (1)

42. Postnotum 3 elevated on membrane: absent (0); present (1).

43. Male cerci: thickened and spatulate (0); thickened at bottom only coming to a tapered tip, forming a horn shape (1)

44. Last three abdominal segments of male contracted so each is more or less of similar length: absent (0); present (1)

45. Viewed dorsally, last three abdominal segments of male expanded in width: absent (0); present (1)

46. Abdominal segment 10 with dorsal surface much longer than ventral surface: absent (0); present (1)

47. Phytotelmata used as larval habitats: absent (0); present (1)

48. Leaf axils of epiphytic bromeliads used as larval habitats: absent (0); present (1)

49. Leaf axils of terricolous bromeliads used as larval habitats: absent (0); present (1)

50. Bamboo used as larval habitats: absent (0); present (1)

51. Fruit husks lying on ground used as larval habitats: absent (0); present (1)

52. Tree holes used as larval habitats: absent (0); present (1)

53. Scutum 3 bisected by deep groove: absent (0); present (1).

54. Shape of tergal apophysis 2: flat or slightly surrounded inward (0); deep triangular notch

(1); deep round notch (2); deep heart shaped notch (3).

55. Antealar Sinus: Deeply notched (0); rounded (1); flat (2).

56. CuA straight: absent (0); present (1).

57. Pterostigmal brace vein forming distal side of triangular cell in forewing: absent (0); present (1). Look to see if only present in male/ forewing

58. Large articulated basal spine on male-superior appendages: absent (0); present (1).

59. Bump in hind wing over pterostigma: absent (0); present (1).

60. Large species: hind wing >50mm in length (make in ref to femur length or abdominal seg.): hind wing < 47 mm in length (0); hind wing > 50 mm in length (1)

APPENDIX III

Character matrix (60 characters) for all 30 taxa included in this study

Lestes disjunctus 00000111111000000111001100100100000000100001000?????12000000

Teinobasis ariel 011112110010000011001111001000000000001101110011000003001100

Teinobasis fortis 01111211001000001100111100100000000000110011000?????03000100

Calopterix aequabilis 00112000111000021011000000101120000000100010000?????11010000

Coenagrion resolutum 00111211001000000100111100100000000000000010000?????03000000

Megalagrion blackburni 00002211001000001000111100100110001000200111100?????13000000

Leptagrion perlongum 012112110010000011001111001100000000001101200010100013000000

Leptagrion macrurum 011112110010000011001111000000000000001111110010100013000000

Diceratobasis melanogaster 012112110010000001001111001?0000000000000100?011000013001000

Pericnemis strictica 11111211001000001100111110?00000001000???1????10010011000000

Pericnemis incallida 11111211001000001100111110110000001000210110000?????11000000

Roppaneura beckeri 012112110010001111001111001000000000011101100010100013010000

Coryphagrion grandis 012012110010000011011111111120000011111101100010001113110001

Anomisma abnorme 11211220101010002011000111112101100111010100001??????1000001

Mecistogaster linearis 012122201010000020001111110101000011111111000010000110200001

Mecistogaster amalia 012122201010000020000111110101000011111011201010000113100011

Mecistogaster astricta 11212220101000002000111111010100001111111100001??1??10000000

Mecistogaster buckleyi 11212220101000002000111111010100001111111101111?????11100000

Mecistogaster jocaste 012122201010000020001111110101000011111111011010000110000000

Mecistogaster lucretia 01212220101000002000111111010100001111111120111?????13100011

Mecistogaster martinezi 1121222010100000200011111101110000111110110011100?0111000000

Mecistogaster modesta 012122201010000020001111110101000011111101100011100013100000

Mecistogaster ornata 112122201010000020001111110101000011111111000010001113100001

Megaloprepus caerulatus 0121212010101001201110001111012111111101010000111101?1000001

Microstigma anomalum 012121201010100020110101111111210111110101100010001011100001

Microstigma maculatum 0121212010101000201101011111012111111101011000100001?1100001

Microstigma rotundatum 012121201010100020110101111111211111110101000010001111100001

Pseudostigma aberrans 012122201010000020110001111101100011111111001110000113100001

Pseudostigma accedens 012122201010000020110001111101100011111101001110000113100001

Telebasis salva 11111211101000000100000001?00000000000???0????0?????10000?0?