(Coleoptera: Glaphyridae) of the Aegean: from Morphologυ to Phylogenυ

UNIVERSITY OF THE AEGEAN DEPARTMENT OF ENVIRONMENT

PYGOPLEURUS (COLEOPTERA: GLAPHYRIDAE) OF THE AEGEAN: FROM MORPHOLOGΥ TO PHYLOGENΥ

Vangelis-Loukas Mizerakis

Advising professor: Triantaphyllos Akriotis (Department of Environment)

Committee Members: Theodora Petanidou (Department of Geography) Konstantinos Theodorou (Department of Environment)

Lesvos, March 2016

ABSTRACT

The genus Pygopleurus belongs to the Glaphyridae family (Coleoptera) and is the main pollinator of the «red anemone guild» flowers. The faunistic composition for these beetles is poorly known for the Aegean islands, as their taxonomy is considered very complex. This study applies some successive research steps in order to clarify the taxonomic, phylogenetic and geographic status of these beetles in the Aegean archipelago.

The beetle specimens studied were collected applying the pantrapping and handnetting methods in a systematic collection on 25 Aegean islands including eastern Aegean (from Thasos to Karpathos) and Central Aegean (Cyclades). All collected specimens were sorted into morphospecies using a dissecting microscope.

The detected morphological differences were pinpointed, photo-illustrated in detail, and used for the generation of a phylogenetic tree based on a “maximum parsimony” analysis. The above morphology procedure led to the detection of 50 characters that can be used for specimen identification. Based on the generated phylogenetic tree, the 5 pre-sorted Pygopleurus morphospecies were clearly separated by their morphological differences and considered as five distinct species. In addition, the analysis showed further differentiations within the species level. A genetic study may lead to the detection of taxa at finer taxonomic level, as well as an additional species (Pygopleurus sp.3). The analysis of the distribution patterns revealed that the Pygopleurus beetles are only present on the East Aegean islands; Lesvos, with five species was the island with the highest species richness among all islands explored.

ABSTRACT (GREEK VERSION)

Το γένορ Pygopleurus ανήκει ζηην οικογένεια Glaphyridae (Κολεόπηεπα) και αποηελεί ηον κύπιο επικονιαζηή ηων «κόκκινων διζκοειδών ανθών». H πανιδική ηοςρ ζύνθεζη ζηα νηζιά ηος Αιγαίος είναι ελάσιζηα μελεηημένη, μιαρ και η ηαξινόμηζή ηοςρ είναι δςζσεπήρ. Η ζςγκεκπιμένη μελέηη έσει ζκοπό να ζςνειζθέπει ζηην ηαξινομική, θςλογενεηική και γεωγπαθική γνώζη ηων ζκαθαπιών αςηών ζηο Αιγαίο.

Μελεηήθηκαν 136 ένηομα πος ζςλλέσθηκαν ζςζηημαηικά με απόση και πινακοπαγίδερ ζε 25 νηζιά ηος Αιγαίος. Τα ζςλλεσθένηα άηομα διασωπίζηηκαν ζε ομάδερ και διακπίθηκαν ζε μοπθοείδη, ζηη βάζη διακπιηών μοπθολογικών ηοςρ σαπακηήπων πος παπαηηπήθηκαν μέζω ζηεπεοζκοπίος. Οι μοπθολογικοί αςηοί σαπακηήπερ, αθος καηαγπάθηκαν και θωηογπαθήθηκαν λεπηομεπώρ, σπηζιμοποιήθηκαν για ηην δημιοςπγία ενόρ θςλογενεηικού δένηπος «μέγιζηηρ θειδωλόηηηαρ» ηων μοπθοειδών.

Η μοπθολογική μελέηη ηων Pygopleurus, οδήγηζε ζηον ενηοπιζμό 50 σαπακηήπων πος μποπούν να σπηζιμοποιηθούν για ηην αναγνώπιζη ηων δειγμάηων ζε επίπεδο είδοςρ ζηο απσιπέλαγορ ηος Αιγαίος. Σηη ζςνέσεια, η καηαζκεςή ηος θςλογενεηικού δένηπος επιβεβαίωζε ηην διάκπιζη ζε πένηε είδη ηος γένοςρ, όπωρ είσαν μοπθολογικά διακπιθεί (5 μοπθο-είδη). Επιπλέον, εμθανίζηηκαν και κάποιερ διαθοποποιήζειρ ενηόρ ηων ειδών, με ενδιαθέποςζα πεπίπηωζη ηο Pygopleurus sp. 3, όπος μια γενεηική ανάλςζη πιθανώρ να οδηγήζει ζηην ςπαπξη ςποείδοςρ, ή και διαθοπεηικού είδοςρ. Ένα ενδιαθέπον ζηοισείο αθοπά ζηον πεπιοπιζμό ηηρ εξάπλωζηρ ηος γένοςρ μόνο ζηα νηζιά ηος αναηολικού Αιγαίος. Η Λέζβορ, με πένηε είδη, διαθέηει ηον μεγαλύηεπο πλούηο ειδών ζε ζσέζη με ηα ςπόλοιπα νηζιά ηος Αιγαίος. Το είδορ Pygopleurus sp. 1 έσει ηην πιο εςπεία εξάπλωζη ζηα νηζιά, ενώ ηο Pygopleurus sp. 2 ηην πιο πεπιοπιζμένη (μόνο ζηην Λέζβο).

KEY WORDS

Glaphyridae, Pygopleurus, Taxonomy, Morphology, Pollination, Aegean islands

TABLE OF CONTENTS

ABSTRACT ...... 2 ABSTRACT (GREEK VERSION) ...... 3 KEY WORDS ...... 4 TABLE OF CONTENTS ...... 5 FIGURES ...... 7 TABLES ...... 7 INTRODUCTION ...... 8 MATERIAL AND METHODS ...... 9 Data-set information ...... 9 Morphological characters ...... 10 Specimens state evaluation ...... 10 Phylogenetic Analysis ...... 11 Species distribution map ...... 11 Species illustration ...... 11 RESULTS ...... 12 Morphological characters ...... 12 Specimen information list ...... 14 Phylogenetic analysis ...... 18 Species distribution map ...... 23 DISCUSION ...... 24 Morphological characters ...... 24 Phylogenetic Analysis ...... 24 Species distribution ...... 25 Morphospecies to species matching ...... 26 To conclude ...... 27 ACKNOWLEDGEMENTS ...... 28 REFERENCES ...... 29 APPENDIX 1. ILLUSTRATION OF MORPHOLOGICAL STATES ...... 30 Body ...... 30

Head parts ...... 30 Clypeus ...... 32 Pronotum ...... 35 Elytra ...... 38 Scutellum ...... 40 Hairs ...... 40 Legs ...... 46 Abdomen ...... 48 Genitalia ...... 49 APPENDIX 2. CHARACTER STATE EVALUATION ...... 52 APPENDIX 3. PHYLOGENETIC TREE ...... 52 APPENDIX 4. SPECIES ILLUSTRATION ...... 52 Pygopleurus foina ...... 52 Pygopleurus kareli ...... 53 Pygopleurus labaumei ...... 53 Pygopleurus vulpes ...... 53 Pygopleurus medius ...... 54

FIGURES

Figure 1. Phylogenetic tree. of the genus Pygopleurus based on Maximum parsimony analysis that generated 6552 equally parsimonious trees. Length=134 steps, Consistency index (CI)=55, Retention index (RI)=96. Filled circles denote unique changes and open circles non-unique changes. Values above circles represent character number and values below, character state. Values above branches indicate Bootstrap values...... 22 Figure 2. Distribution map for the Pygopleurus morphospecies. Grey colored areas represent the sampled areas, whereas each symbol represents the distribution of a morphospecies...... 23

TABLES

Table 1. Description of the year, sampling method and number of sites for each of the sampled Aegean islands. Melissotheque of the Aegean (Petanidou et al. unpublished data) ...... 9 Table 2 The detected morphological characters and their states. Characters and character states for outgroup beetles are marked with asterisk symbol...... 12 Table 3 Information list of the selected and sorted in gender and morphospecies groups, Pygopleurus and the identified and sorted in gender outgroup specimens (Eulasia pareyssei). The specimen code number refers to the accession number of the Melissoteque of the Aegean (Petanidou et al. 2013)...... 14

INTRODUCTION

Pygopleurus (synonym: Amphicoma) beetles belong to the family “Glaphyridae” of the “Coleoptera” order. They are strong and active during the day fliers, mostly brightly colored and hairy, often with markings and coloration resembling bees and bumblebees. Mainly they are observed flying and hovering near flowers, foliage and sandy areas (Keasar et al. 2010) and in the Eastern Mediterranean regions they are one of the main plant pollinators during springtime (Bollino & Ruzzante 2015). Specifically the species of Pygopleurus and Eulasia (Glaphyridae) are the primary pollinators of red anemone guild flowers (species of genera: Anemone, Tulipa, Ranunculus, Adonis, Glaucium and Papaver), sharing an impressive symbiotic relationship (Keasar et al. 2010; Dafni et al. 1990). They feed on floral pollen and mate on flowers, without consuming and harming ovaries and petals, as other Glaphyridae beetles do and also regularly shelter at night within the closed flowers (Keasar et al. 2010) .The red flower color appears to have an innate attraction effect on them (Dafni A. 1997), whereas flower size and black-red flower’s contrast is increasing the attraction level (Keasar et al. 2010; Dafni et al. 1990). These facts are indicating a strong co-evolutionary relationship between those beetles and the “red anemone guild” (Keasar et al. 2010). Glaphyridae is considered a big family, as it includes approximately 190 species in Palearctic region, with the highest species diversity in Asia Minor (Turkey), and the Near East, which is therefore known as a centre of speciation (Keasar et al. 2010). Although Pygopleurus genus and Glaphyridae family is in general a beetle taxa with a wide abundance and ease of sampling (Bollino & Ruzzante 2015), there is an important lack of phylogenetic studies (Keasar et al. 2010) and the Aegean islands faunistic composition is poorly known (Bollino & Ruzzante 2015). These facts, in addition with the big proposed synonymies number, the ancient species rehabilitation (Baraud 1989) and the unreliability and diversity of their external characters (Keith & Uliana 2008) makes their taxonomy and systematics a hard and challenging taxa group to deal with. In order to solve these difficulties, regarding the Pygopleurus fauna of the Aegean islands, this study applies the following successive researching steps. Firstly, it detects and records many morphological characteristics that lead to a morphospecies determination. Then it creates a phylogenetic tree, based on the differentiation of the beetles and finally, in consideration of morphological and geographical information, it matches the morphospecies to the respective species names.

MATERIAL AND METHODS

Data-set information The beetle specimens have been collected from the Aegean islands within the frame of POL- AEGIS and the ALARM project and were stored at the Melissotheque of the Aegean reference collection in the Biogeography and Ecology Laboratory of the Aegean University. The sampling has been carried out from late March to early July of the years: 2004 (ALARM), 2012, 2013, 2014 and 2015 (POL-AEGIS) with the same, Europe-wide tested, protocol. Specifically, the sampling method was mainly based on the handnetting procedure (for plant–pollinator relationships record) and less on the pantraping (for systematic pollinator diversity assessment). Each site was sampled in three rounds per year (Petanidou et al. 2013, Lazaro et al. 2016). On Table 1 below, you can see details on the samplings per each island.

Table 1. Description of the year, sampling method and number of sites for each of the sampled Aegean islands. Melissotheque of the Aegean (Petanidou et al. unpublished data)

Island Year Method # of Sites Anafi 2013 hand-netting 3 Antikythera 2014 no systematic sampling 9 Chios 2012 - 2015 hand-netting; pantrapping (coleoptera occasionally kept) 30 Delos 2013, 2015 hand-netting 1 Folegandros 2014 hand-netting 5 Ikaria 2012 hand-netting 6 Ios 2013 hand-netting 14 Irakleia 2013, 2014 hand-netting 7 Karpathos 2012 hand-netting 9 Kea 2013 hand-netting 12 Kos 2012 hand-netting 6 Kythnos 2015 hand-netting 8 Lesvos 2004 - 2009 & 2011 - 2015 mainly hand-netting; pantrapping only at years 2011 & 2012 20 Limnos 2012 hand-netting; pantrapping only at antennae sites 21 Milos 2013 hand-netting 3 Mykonos 2013, 2015 hand-netting 3 Naxos 2012 hand-netting 13 Paros 2013 hand-netting 4 Rhodos 2012 hand-netting; pantrapping 29 Samothrace 2012 hand-netting 5 Santorini 2013 hand-netting 4 Serifos 2013, 2015 hand-netting 9 Sifnos 2011 only occasional sampling 4 Syros 2013 hand-netting 5 Thasos 2012 hand-netting 8 Tinos 2014 hand-netting 7

Morphological characters Pygopleurus is considered a taxonomically demanding genus. In fact, there are no taxonomical keys, reference collection available, and identification literature in other language but French; moreover, the absence of reliable external identification characters makes the effort much more difficult (Keith & Uliana 2008). Many attempts to identify the species with the French Jacques Baraud identification key (1992) took place, but none was efficient. To overcome all these taxonomical difficulties of the genus, all specimens have been examined by stereo microscope for a long time, with an extensive focus on their external morphological characteristics. Males and females were sorted separately and the genitalia of the male beetles have been extracted, as this character is considered the most reliable separating factor (Keith & Uliana 2008). Then the male beetles have been sorted to morphospecies, according to the similarity of the extracted genitals, and have been compared for further differing morphological characteristics. Some of the detected characteristics could be also applied for the female specimens, not only to sort them to morphospecies too, but also to match them with their respective male morphospecies. In order to implement the Phylogenetic analysis, all these Pygopleurus (Glaphyridae) specimens have also been compared morphologically with an outgroup species (Eulasia pareyssei, (Glaphyridae)). Every characteristic of the body bearing differentiations, was described as a “character” (e.g. “antennae color”) and the differing characteristics (changes) of a character were described as “states” (e.g. “0=black, 1=blackened, 2=almost no black”). Some states of the character, “Pronotum (b) color (16b)” have been sorted in color groups and a new character was created, “Pronotum color (16)”, as they were not functional for some beetle morphospecies (individuals of different species often had the same color, whereas individuals inside of the same species had different colors). It has to be noticed, that some of the used characters are also found in the Baraud identification key (1989), but others are possibly new for science. All these characters with their states, are presented in the Table 2, of the results. For a more detailed description, there has been created Appendix 1, with photo illustrations of the character states. (taken by stereo microscope camera “Jenoptik, ProgRes SpeedXT core 5”)

Specimens state evaluation In order to prepare the input files for the phylogenetic analysis, 116 Pygopleurus (Glaphyridae) beetles, and 20 Eulasia pareyssei (Glaphyridae) outgroup beetles have been examined. Pygopleurus specimens were from Limnos, Lesvos, Chios, Ikaria, Kos, Rhodos and outgroup specimens from Limnos, Lesvos and Thermi (Thessaloniki). There has been chosen a number of at least 20 beetles (males and females in equal proportion) per sorted morphospecies group with specimens from all island populations. All the selected specimens are presented below in Table 3. Every single specimen has been examined for each of the recorded characters of Table 2 and evaluated with a state value. The male-specific characters have not been examined for female specimens and the female-specific have not been examined for male specimens. The character “50” and the “16b” although examined, had not been included for the input files for the phylogenetic analysis, as its states did not contribute to the resolution of the resolution of the

10 tree. All the specimen’s character evaluation have been recorded and published online (Appendix 2).

(https://docs.google.com/spreadsheets/d/1499PPSKlN6bfQx-vTtigIzSdlqViBra4XwsfUDzs- Uo/pub?gid=155573365&single=true&output=pdf)

Phylogenetic Analysis The data was analysed with the heuristics search strategy “Maximum parsimony” (MP). The MP analysis was performed in the program NONA (Goloboff 1999), spawned in WINCLADA version 1.00.08 (Nixon 2002). A heuristics search algorithm with 1000 random addition replicates (mult x 1000) was performed with holding 10 trees per round (hold ⁄ 10), max trees set to 10 000 and applying TBR branch swapping. All input files for NONA were generated within the Mesquite 2.75 system for phylogenetic computing (Maddison & Maddison 2011). The phylogenetic tree is illustrated in Figure 1 of the results and linked online in Appendix 3.

(https://drive.google.com/file/d/0Bww89sOaYEypSE10ak9wYi1Sd0U/view?usp=sharing)

Species distribution map After the statistical verification of the phylogenetic analysis for the morphospecies differentiation of the Pygoleurus specimens, a Map of the Aegean islands with the sampled areas and the distribution area for each morphospecies has been created, with “simplemappr” online program (http://www.simplemappr.net/). The map, is illustrated in Figure 2.

Species illustration At last, both gender of each species has been photographed with a digital camera and illustrated in Appendix 4. To each species photos has also been added an illustration of all the possible species colors.

RESULTS

Morphological characters In the table 2 below, are presented all the detected morphological characters and states. In the first column is the character numbering mentioned, in the second we have the beetle body parts with a description of the examined character in the third column and in the last we have a description of all the possible states of the characters. Character states with asterisk symbolize outgroup beetles changes. If the asterisk is also mentioned in the character description column, then these characters represent outgroup differences with the whole Pygopleurus genus.

Table 2 The detected morphological characters and their states. Characters and character states for outgroup beetles are marked with asterisk symbol.

Char. Body part Character description Character state # 0 Body Coloration * 0=Abdomen metallic black, thorax and legs (2nd,3rd) metallic green, 1=All body (excl. front legs) metallic light green* 1 Head parts Last maxillary palp segment size ratio to 0=last smaller*, 1=+/-same length, 2=last bigger the two segments before 2 Groove at last segment of maxillary palp 0= wide and deep*, 1= thin and deep, 2=wide and flat, 3=wide and deeply flattened 3 Internal color of 3 last antennae segments 0=black*, 1=blackened, 2=almost no black 4 Antennae sensor structure (top of last 0= two parallel grooves, 1=one horseshoe shaped segment) * groove* 5 Eyes cavity size* 0=narrow*, 1=very narrow 6 Clypeus With glossy bald surface or area with very 0= No, 1=Yes* sparse punctuation * 7 Anterior shape 0= round*, 1=rectangular, 2=rounded angle 8 Anterio-lateral edges with strongly 0= No, 1=Yes* elevated peak * 9 Middle keel state 0=absent, 1=present, 2=flat 10 Punctuation (only males) 0=sparse, 1=dense, 2=mixed dense and very sparse* 11 Pronotum Posterio-lateral with 2 glossy bald/ low 0=No, 1=Yes* punctuation dense surfaces * 12 Punctuation 0=sparsely, 1=dense 13 Vermiculation 0=absent, 1=irregular, 2=straight 14 Posterio-lateral edges 0=rounded, 1=rounded angle, 2=angled 15 Medio-apical edge curved apicaly * 0= No, 1=Yes* 16 Color 0=diverse mix (dark-green, green, yellow-green, purple, petrol, blue, orange-bronze, red-bronze, greenish red- bronze) 1=purple-burgundy, 2=light-green* 17 Glossiness 0=matt, 1=glossy 18 Elytra Apex of the elytra strongly remoted * 0= No, 1=Yes* 19 Thickness (lateral view) * 0=flat, 1=high-thick * 20 Apex edge angle 0=rounded, no angle* 1=vertical, 2=outwards,

3=inwards 21 Apex with spike 0= No, 1=Yes 22 Posteriorly black (of females) 0= No, 1=Yes 23 Scutellum Shape * 0=triangular, 1=short-rounded* 24 Hairs Black and white elytral pattern * 0= No, 1=Yes* 25 Black elytral bristles length* 0=long*, 1=short 26 White haired body with orange haired 0= No, 1=Yes* abdomen* 27 Hair at last tergite (of females) * 0=short*, 1=long 28 Very Intense orange hair all over body 0= No, 1=Yes 29 Elytral bristles at apex edge 0=week, 1=intense 30 Black bristles at elytra surface (excl. edge) 0=absent, 1=thin, 2=thick, 3=thin and thick mixed 31 Small lying elytral hair 0=absent, 1=thin, 2=thick 32 White-orange elytral bristles 0=absent, 1=present, 2=present, but different* 33 White long and thin elytral hair * 0=absent, 1=present* 34 White elytral bristles only at the inside 0=No, 1=Yes* edge * 35 Black bristles intense all over elytra surface 0=No, 1=Yes 36 Lateral sternite hair 0=black (orange/white only at top), 1=white-orange, 2=mixed white/orange with black, 3=mixed small black with white spikes/hairs, 4= long thin white-orange* 37 Legs Mesotibia and metatibia covered with thick 0=Yes, 1=No* spikes * 38 Apical spurs of metatibia color* 0=half black half white 1=black with brown tip*, 39 Bristels at apical part of protibia color 0=black, 1=dark brown, 2=very light brown 40 Protibia color 0=metallic green, 1=metallic dark colors 41 Nails of front leg 0=almost not curved* 1=lightly curved, 2=curved, 3=strongly curved 42 Abdomen Last abdominal segments red-brown (of 0=Yes, 1=No* males) * 43 Last abdominal segment big and strongly 0= Yes, 1=No* protrudes downwards (of males) * 44 5th sternite very enlarged * 0= Yes, 1=No* 45 Sternites V shaped (of females) * 0= No*, 1=Yes 46 Last tergite size (of females) * 0=length +/- equal to width, 1=length

Specimen information list In the Table 3 below, is presented a detailed list of information for the selected specimens. Information is listed with the following series: Specimen code number, Region, Island/Municipality, Site, Collection date and identified Genus, Species/Morphospecies and Gender.

Table 3 Information list of the selected and sorted in gender and morphospecies groups, Pygopleurus and the identified and sorted in gender outgroup specimens (Eulasia pareyssei). The specimen code number refers to the accession number of the Melissoteque of the Aegean (Petanidou et al. 2013).

Specimen Region Island/ Site Collection date Genus Species/ Gender code Municipality Morphospecies number 030704 Aegean Islands Limnos Katalakkos 08/05/2012 Eulasia pareyssei male 030698 Aegean Islands Limnos Katalakkos 08/05/2012 Eulasia pareyssei male 030692 Aegean Islands Limnos Katalakkos 08/05/2012 Eulasia pareyssei male 001438 Aegean Islands Limnos Sand Dunes 10-12/05/2012 Eulasia pareyssei male 030699 Aegean Islands Limnos Katalakkos 08/05/2012 Eulasia pareyssei male 031250 Aegean Islands Limnos Katalakkos 08/05/2012 Eulasia pareyssei male 030710 Aegean Islands Limnos Katalakkos 08/05/2012 Eulasia pareyssei male 095486 Thessaloniki Thermi site 2 15/05/2013 Eulasia pareyssei male 095461 Thessaloniki Thermi site 1 15/05/2013 Eulasia pareyssei male 095497 Thessaloniki Thermi site 3 16/05/2013 Eulasia pareyssei male 003322 Aegean Islands Lesvos Karava 11-13/04/2012 Eulasia pareyssei female 003200 Aegean Islands Lesvos Karava 11-13/04/2012 Eulasia pareyssei female 003176 Aegean Islands Lesvos Karava 11-13/04/2012 Eulasia pareyssei female 003257 Aegean Islands Lesvos Karava 11-13/04/2012 Eulasia pareyssei female 003258 Aegean Islands Lesvos Karava 11-13/04/2012 Eulasia pareyssei female 152301 Aegean Islands Lesvos Karava 11-13/04/2012 Eulasia pareyssei female 095483 Thessaloniki Thermi site 2 15/05/2013 Eulasia pareyssei female 095460 Thessaloniki Thermi site 1 15/05/2013 Eulasia pareyssei female 095472 Thessaloniki Thermi site 1 15/05/2013 Eulasia pareyssei female 010941 Aegean Islands Limnos Thanos 02-04/05/2012 Eulasia pareyssei female 012232 Aegean Islands Lesvos Sigri 4 29/04/2012 Pygopleurus sp.1 male 012450 Aegean Islands Lesvos Vatousa 1 12/05/2012 Pygopleurus sp.1 male 003165 Aegean Islands Lesvos Karava 11-13/04/2012 Pygopleurus sp.1 male 003234 Aegean Islands Lesvos Karava 11-13/04/2012 Pygopleurus sp.1 male 012098 Aegean Islands Lesvos Vatousa 1 09/04/2012 Pygopleurus sp.1 male 036151 Aegean Islands Rhodos Fletrovouno 19-21/03/2012 Pygopleurus sp.1 male Laermon_6.4 027129 Aegean Islands Rhodos Fragma Apolakkias_M5 20-22/03/2012 Pygopleurus sp.1 male 046532 Aegean Islands Rhodos Agros-Profylia_4.1 24-26/04/2012 Pygopleurus sp.1 male 027137 Aegean Islands Rhodos Fragma Apolakkias_M5 20-22/03/2012 Pygopleurus sp.1 male 046639 Aegean Islands Rhodos Axla Asklipeiou_4.2 24-26/04/2012 Pygopleurus sp.1 male

037595 Aegean Islands Chios Kato Fana 15-16/04/2012 Pygopleurus sp.1 male 071475 Aegean Islands Chios Gridia 12/04/2013 Pygopleurus sp.1 male 110116 Aegean Islands Chios Emborios 02/04/2014 Pygopleurus sp.1 male 036466 Aegean Islands Kos Psallidi 07/04/2012 Pygopleurus sp.1 male 017846 Aegean Islands Kos Kyparissodassos 06-08/04/2012 Pygopleurus sp.1 male 036430 Aegean Islands Kos Asklipieion 06/04/2012 Pygopleurus sp.1 male 035219 Aegean Islands Ikaria Proespera 19/04/2012 Pygopleurus sp.1 male 035368 Aegean Islands Ikaria Evdilos 23/04/2012 Pygopleurus sp.1 male 003743 Aegean Islands Lesvos Aspropotamos 11-13/04/2012 Pygopleurus sp.1 female 003887 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.1 female 003902 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.1 female 003209 Aegean Islands Lesvos Karava 11-13/04/2012 Pygopleurus sp.1 female 003032 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.1 female 032229 Aegean Islands Rhodos Ahla Asklipeiou_4.4 20-22/03/2012 Pygopleurus sp.1 female 027237 Aegean Islands Rhodos Kouara Laermon_1.2 20-22/03/2012 Pygopleurus sp.1 female 046704 Aegean Islands Rhodos Daskalou Asklipeiou_4.3 24-26/04/2012 Pygopleurus sp.1 female 046903 Aegean Islands Rhodos Stifi-Profylia_ 24-26/04/2012 Pygopleurus sp.1 female 5.3 046641 Aegean Islands Rhodos Axla Asklipeiou_4.2 24-26/04/2012 Pygopleurus sp.1 female 114696 Aegean Islands Chios Kato Fana 30/04/2014 Pygopleurus sp.1 female 037811 Aegean Islands Chios Gridia 10/04/2012 Pygopleurus sp.1 female 037563 Aegean Islands Chios Kato Fana 15-16/04/2012 Pygopleurus sp.1 female 034268 Aegean Islands Kos Kastro 09/04/2012 Pygopleurus sp.1 female Antimachias 036463 Aegean Islands Kos Psallidi 07/04/2012 Pygopleurus sp.1 female 036472 Aegean Islands Kos Psallidi 07/04/2012 Pygopleurus sp.1 female 017486 Aegean Islands Kos Ag. Theologos 08-10/04/2012 Pygopleurus sp.1 female 035225 Aegean Islands Ikaria Proespera 19/04/2012 Pygopleurus sp.1 female 004983 Aegean Islands Lesvos Pigi 05-07/05/2012 Pygopleurus sp.2 male 002926 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 002948 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 003021 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 152300 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 002930 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 002993 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 002991 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 002996 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 002947 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 male 004461 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 004453 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 004412 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 004423 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 004413 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female

003076 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 female 004462 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 004415 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 003014 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.2 female 004472 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.2 female 038264 Aegean Islands Chios Aghio Gala 19/04/2012 Pygopleurus sp.3 male 038184 Aegean Islands Chios Managros 29/03/2012 Pygopleurus sp.3 male 026471 Aegean Islands Chios Aghio Gala 31/03- Pygopleurus sp.3 male 02/04/2012 026664 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 male 026660 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 male 026461 Aegean Islands Chios Aghio Gala 31/03- Pygopleurus sp.3 male 02/04/2012 026659 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 male 004381 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.3 male 004398 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.3 male 004368 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.3 male 038180 Aegean Islands Chios Managros 29/03/2012 Pygopleurus sp.3 female 038261 Aegean Islands Chios Aghio Gala 19/04/2012 Pygopleurus sp.3 female 026697 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 female 026587 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 female 026584 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 female 026632 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 female 026698 Aegean Islands Chios Managros 29-30/03/2012 Pygopleurus sp.3 female 004445 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.3 female 004378 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.3 female 004392 Aegean Islands Lesvos Pigi 12-14/04/2012 Pygopleurus sp.3 female 030733 Aegean Islands Limnos Phalakros 06/04/2012 Pygopleurus sp.4 male 002159 Aegean Islands Limnos Thanos 05-07/04/2012 Pygopleurus sp.4 male 031133 Aegean Islands Limnos Katalakkos 11/04/2012 Pygopleurus sp.4 male 031144 Aegean Islands Limnos Katalakkos 11/04/2012 Pygopleurus sp.4 male 022978 Aegean Islands Limnos Kotsinas 06-08/04/2012 Pygopleurus sp.4 male 011856 Aegean Islands Lesvos Sigri 3 03/04/2012 Pygopleurus sp.4 male 011857 Aegean Islands Lesvos Sigri 3 03/04/2012 Pygopleurus sp.4 male 003854 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.4 male 003837 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.4 male 004239 Aegean Islands Lesvos Parakoila 11-13/04/2012 Pygopleurus sp.4 male 030727 Aegean Islands Limnos Phalakros 06/04/2012 Pygopleurus sp.4 female 001367 Aegean Islands Limnos Katalakkos 07-09/04/2012 Pygopleurus sp.4 female 031153 Aegean Islands Limnos Katalakkos 11/04/2012 Pygopleurus sp.4 female 015890 Aegean Islands Limnos Moudros II 04-06/04/2012 Pygopleurus sp.4 female village 013481 Aegean Islands Limnos Plaka - Panagia 03-05/04/2012 Pygopleurus sp.4 female

011855 Aegean Islands Lesvos Sigri 3 03-03/04/2012 Pygopleurus sp.4 female 003848 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.4 female 003872 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.4 female 003906 Aegean Islands Lesvos Kalloni - Ag.Paraskevi 11-13/04/2012 Pygopleurus sp.4 female 004196 Aegean Islands Lesvos Parakoila 11-13/04/2012 Pygopleurus sp.4 female 0000408 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 male 0000325 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 male 0000419 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 male 0007905 Aegean Islands Lesvos Pigi 26-28/03/2004 Pygopleurus sp.5 male 0015729 Aegean Islands Lesvos Pigi 26-28/03/2004 Pygopleurus sp.5 male 0015326 Aegean Islands Lesvos Mytilene 06/04/2004 Pygopleurus sp.5 male 0000576 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 male 0000579 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 male 0000355 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 male 0000955 Aegean Islands Lesvos Loutra 26-28/03/2004 Pygopleurus sp.5 male 0015325 Aegean Islands Lesvos Mytilene 06/04/2004 Pygopleurus sp.5 female 0015784 Aegean Islands Lesvos Loutra 26-28/03/2004 Pygopleurus sp.5 female 0000391 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 female 0000566 Aegean Islands Lesvos Mytilene 22-24/03/2004 Pygopleurus sp.5 female 003114 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.5 female 003279 Aegean Islands Lesvos Karava 11-13/04/2012 Pygopleurus sp.5 female 002998 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.5 female 003083 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.5 female 003136 Aegean Islands Lesvos N Kydonies 11-13/04/2012 Pygopleurus sp.5 female 003308 Aegean Islands Lesvos Karava 11-13/04/2012 Pygopleurus sp.5 female

Phylogenetic analysis In the next pages, in Figure 1 (split in 4 pages) is the illustration of the maximum parsimony analysis created Phylogenetic tree of the Pygopleurus genus specimens. The analysis generated 6552 equally parsimonious trees, with tree Length=134 steps, Consistency index (CI)=55, and Retention index (RI)=96. Filled circles denote unique changes and open circles non-unique changes. The values above circles represent character number and the values below circles represent character state. Bootstrap values, are indicated above branches, ranging from 88 to 100.

(For a total view of the phylogenetic tree, in pdf file, click the following link: https://drive.google.com/file/d/0Bww89sOaYEypaGgzb0J5S0wwd3M/view?usp=sharing)

Species distribution map The distribution map of the Pygopleurus morphospecies in the Aegean archipelago is illustrated in Figure 2. All the sampled areas during the POL-AEGIS and ALARM project are grey colored and each morphospecies distribution is marked with a symbol.

Figure 2. Distribution map for the Pygopleurus morphospecies. Grey colored areas represent the sampled areas, whereas each symbol represents the distribution of a morphospecies.

DISCUSION

Morphological characters In total, as we see in Table 2 of the results, 51 characters (+ old color state’s version “16b”) were recorded, of which 27 regard differences within the Pygopleurus genus and 24 differences of Pygopleurus genus with the outgroup. Due to the character state evaluation of the specimens in Appendix 2 we can sort the examined characters in 39 gender-independent characters, 7 male and 5 female characters. The gender-independent can be sorted further, in 32 qualitative (strong) characters, 7 non qualitative (week) characters. A character is named qualitative, when it provides stable character state values for all individuals and for both gender inside each species group. Qualitative characters can be used reliably for species sorting and offer a good possibility for the creation of a Pygopleurus identification key. They can also be used without knowledge of the specimen gender, something helpful for eg. in the case of Pygopleurus sp.4. Pygopleurus sp.4 has a strong sexual dimorphism (males are completely orange colored, whereas females are greenish and brown) and could be easily mistaken as different species. The male and female characters, are and can be used also as qualitative characters, in case where specimens are gender identified. Character number 31 (“Small lying elytral hair”) should be used with caution. It is qualitative for all specimens except of Pygopleurus sp.4, of which males and females have different states. It has to be noticed, that the pronotum color (16b) of these beetles can be very misleading, as it provides no stable state values for most of the species. If so, it should be examined in color groups, as in character number 16. Only the purple-burgundy color and light- green can lead to a trusted identification, and with caution the orange and red-bronze, but only for male specimens. At last, character number 50 (“# of toothings at basal part of last protarsus segment (of males)”), does not provide any stable state values per morphospecies, and should be avoided.

Phylogenetic Analysis The phylogenetic analysis not only produced a phylogenetic tree (Figure 1) with a clear branching in the expected five Pygopleurus species, but also lead to a significant statistical support (bootstrap percentage) with very high values (at least 91%) for these species-branches. These facts, prove the existence of at least 5 Pygopleurus species in the Aegean archipelago, but also verifiy the taxinomical effectiveness of the detected morphological Pygopleurus genus characteristics. Additionaly, in many species, we can clearly see the branch separation of the male and female specimens, something expected, given the fact of the observed sexual morphological dimorphism. Very interesting is the fact of the biogeographical morphological differentiation of some species. In Pygopleurus sp.3 we can notice a morphological differentiation between Chios and Lesvos islands specimens, in both of the gender and in Pygopleurus sp.4 between Lesvos and Limnos island specimens. Similarly, in some specimens of Pygopleurus sp.1 we notice small differences between the islands Lesvos, Rhodos, Chios, Kos and Ikaria. We can also notice an morphological differentiation for the Pygopleurus sp.2 and sp. 5 specimens, but inside the same island and with no geographical pattern, as differences are

24 noticed randomly, even within the same sampling sites. It would be worth testing these specimens also with a phylogenetic analysis based on genetical differences, especially for the Pygopleurus sp. 3. It not only has morphological differentiation between its two distribution islands for both gender, but additionally appears to have a different male genital type. Differences in this character are quite important, as genitals are considered a primary identification factor for the Pygopleurus genus (Keith & Uliana, 2008), and could possibly lead to a subspecies or even different species level categorization.

Species distribution Near East and Asia Minor (Turkey), due to the high species richness of the Glaphyridae beetles is considered a centre of speciation for the Pygopleurus genus. (Keasar, et al., 2010) This fact is expected to have a strong influence on the Aegean island distribution of these beetles.

The first thing to notice when observing the map (Figure 2), is that from all sampled areas, Pygopleurus beetles, appear only at the Eastern Aegean islands, and to no other Aegan island. This observation should not be overlooked, given the fact of the same sampling protocol used for all Aegean islands (Petanidou et al. 2013). The formation of the mid-Aegean, a major palaeogeographical event in the geographical history of the Aegean, can strongly support this phenomenon. The mid-Aegean trench is not only separating geologically the Eastern Aegean islands from the Cyclades but is also acting as a biogeographic barrier for reptile, snail, scorpion, isopod and beetle species (Papadopoulou et al. 2009). So, Pyfopleurus species possibly never managed to reach the Cyclades.

The second remarkable fact, is the species richness pattern within the Eastern Aegean islands. We see that in island Lesvos there are found all the five Pygopleurus species, in Chios two, and in Limnos, Ikaria, Kos and Samos only one. That phenomenon can be interpreted with two fundamendal “laws” in ecology. Firstly with the pattern of increasing species richness with increasing surface area (Juergen 2009) for all islands except of Rhodos (Lesvos and Chios are bigger than Limnos, Ikaria and Kos). Secondly with the pattern of increasing species richness with decreasing distance to the nearest mainland (Vujic et al. 2016) for all islands except of Kos (Lesvos and Chios are closer to Asia Minor than Limnos, Ikaria and Rhodos). In addition, the high species richness of Lesvos, can be further supported by the high neighboring surface of the Asia Minor coast line, as Asia Minor is enfolding almost the half of the island’s surface.

At last, we can also come to some conclusions about the species abundance. We can see that Pygopleurus sp.1 is the most widespread species in the Aegean, as it is found in all islands except of Limnos. Pygopleurus sp.3 and sp.4 are middle widespread, as sp.3 exists in Lesvos and Chios and sp.4 in Lesvos and Limnos. In contrast with these species, Pygopleurus sp.2 and sp.5 are only found in Lesvos, which means either they are endemic for Lesvos, either their origin is Near East or Asia Minor, but have no high dispersal abilities in contrast with the other species.

Morphospecies to species matching Many of these study findings, in consideration with a Pygopleurus study report of Bollino & Ruzzante (2015) in Lesvos, allowed a promising morphospecies to species matching. This report not only verifies the existence of five Pygopleurus species on the island, which they have species identified, but additional, provides some helpful information, to achieve this species matching. By knowing the Pygopleurus species fauna of Lesvos we also know the Pygopleurus species fauna composition of all the Aegean.

Pygopleurus sp.5 -> Pygopleurus medius (Petrovitz, 1957)

The unique qualitative character state for Pygopleurus sp.5 specimens: purple-burgundy color of pronotum (character 16, state 1) and posteriorly black elytras of females (character 22, state 1), matched perfectly with the illustrated Pygopleurus medius of the report.

Pygopleurus sp.4 -> Pygopleurus vulpes (Fabricius, 1781)

The unique qualitative character state for the Pygopleurus sp.4 specimens: intensive orange hairs all over body (character 28, state 1) and the character state of black elytral bristles absence (character 30, state 0) matched perfectly with the illustrated Pygopleurus vulpes of the report.

Pygopleurus sp.3 -> Pygopleurus labaumei (Petrovitz, 1971)

The report is using two identification characters proposed by Baraud (1989) for the Pygopleurus labaumei identification: Firstly the “truncated elytral apex” for males which is actually the same character with the elytral character state of Pygopleurus sp.3 and sp.5 of inwards angled apex edge (character 20, state 3) (the inwards angled apex is making the elytral apex to look like cut). Secondly the “well marked elytral sutural tooth” of the females, which in our case is the character of the elytral apex with spike (character 21, state 1) that is also present in Pygopleurus sp.3 and sp.5. But the fact that Pygopleurus sp.5 has the unique character state of purple- burgundy pronotum color and is already matched to Pygopleurus medius, makes Pygopleurus sp.3 the only option for Pygopleurus labaumei.

Pygopleurus sp.2 -> Pygopleurus kareli (Petrovitz, 1962) and

Pygopleurus sp.1 -> Pygopleurus foina (Reitter, 1890)

Two of our examined characteristics that do separate Pygopleurus sp.1 from Pygopleurus sp.2, can help us to match the two remaining species of the report. Pygopleurus sp.1 has intense black bristles all over elytra surface and at the apex edge (character 35, state 1 and character 29, state 1) while Pygopleurus sp.2 has only few and week bristles at elytra syfrace and apex edge (character 35, state 0 and character 29, state 0). As we can see in the species illustration of the report, Pygopleurus kareli has much more week elytral bristles than the remaining Pygopleurus foina. Additionally the collection of Pygopleurus kareli specimens in Lesvos, by Bollino & Ruzzante (2015) was the first report of the species existence in the Aegean and the European

26 fauna, a very compatible fact with the Pygopleurus sp.2 distribution data of our study, restricted at the Lesvos island and found nowhere else. On the other hand, Pygopleurus foina is considered a common species (Bollino & Ruzzante, 2015) something matching very good with the most widespread species of our study, Pygopleurus sp.1.

To conclude The morphological analysis, lead to 32 qualitative characters that can be used effectively for species identification and allows a species identification key creation for the Aegean Pygopleurus fauna. At next, the very high statistical support values of the phylogenetic analysis verify the excistence of at least five Pygopleurus species in the Aegean islands. The phylogenetic tree additionally shows morphological differentiations for some species, like the Pygopleurus labaumei (sp.3) differentiation between Chios and Lesvos islands and a genetic analysis could possibly lead to the finding of new/different taxa. The branching between males and females of the same species reveals also the strong sexual dimorphism Pygopleurus species may have. From the species distribution map, at first we conclude that Pygopleurus species are found only at the Eastern Aegean islands, and possibly never reached the Cyclades. Secondly, we see that island Lesvos has the highest species richness, having all the five Pygopleurus species, and Limnos, Ikaria, Kos and Samos have the lowest, with only one species. At last, we conclude that Pygopleurus foina (sp.1) is the most widespread species in the Aegean, as found almost in all Eastern Aegean islands and Pygopleurus kareli (sp.2) & Pygopleurus medius (sp.5) are the least widespread species, as found only on Lesvos.

ACKNOWLEDGEMENTS

This research has been carried out in the Laboratory of Biogeography & Ecology, Department of Geography, University of the Aegean, in the frame of the POL-AEGIS project (“the POLlinators of the AEGean: dIversity and threatS, 2012-2015”) financed by the Research Funding Program THALES (co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF). Special thanks to T. Petanidou, coordinator of the POL-AEGIS project, for providing the material and the background for this scientific research, to A. Trichas, coleopterologist of the Natural History Museum of Crete, for advising me on many aspects of my thesis work, A. Chroni, PhD student at the Laboratory of Biogeography & Ecology, for supporting me at the phylogenetic tree creation, and T. Tscheulin, lecturer of the Biogeography & Ecology Laboratory for his support on various technical aspects.

REFERENCES Baraud, J. (1989). Révision du genre Pygopleurus Motschulsky (Coleoptera: Scarabaeoidea: Glaphyridae). Annales de la Société Entomologique de France, 25 (3), 331-375.

Bollino, M., & Ruzzante, G. (2015). Corological notes on some species of Pygopleurus (Coleoptera: Glaphyridae) from the Greek Island of Lesbos. Munis Entomology & Zoology, 10, 69-74.

Dafni, A. (1997). The response of amphicoma spp. (Coleoptera; Glaphyridae) beetles to red models differing in area, shape and symmetry. Israel Journal of Plant Sciences, 45:2-3, 247-254.

Dafni, A., Bernhardt, P., Shmida, A., Ivri, Y., Greenbaum, S., O'Toole, C., et al. (1990). Red bowl-shaped flowers: Convergence for beetle pollination in the mediterranean region. Israel journal of botany, 39, 81-92.

Goloboff, P. (1999). NONA (NO NAME), version2. Published by the author.

Juergen, D. (2009). Which function describes the species area relationship best? A review and empirical evaluation. Journal of Biogeography, 36, 728–744.

Keasar, T., Harari, A. R., Guido, S., Keith, D., Dafni, A., Shavit, O., et al. (2010). Red anemone guild flowers as focal places for mating. Biological Journal of the Linnean Society, 99, 808–817.

Keith, D., & Uliana, M. (2008). Contribution to the knowledge of Iranian Pygopleurus Motschulsky, with description of P. rapuzzii n. sp. (Coleoptera: Scarabaeoidea: Glaphyridae). Zootaxa, 1825, 59–64.

Lazaro, A., Tsceulin, T., Devalez, J., Nakas, G., & Petanidou, T. (2016). Effects of grazing intensity on pollinator abundance and diversity, and on pollination services. Ecological Entomology.

Maddison, W. P., & Maddison, D. (2011). Mesquite: a modular system for evolutionary analysis. Version 3.03. http://mesquiteproject.org.

Nixon, K. C. (2002). WINCLADA version 1.00.08. Published by the author.

Papadopoulou, A., Anastasiou, I., Keskin, B., & Vogler, A. P. (2009). Comparative phylogeography of tenebrionid beetles in the Aegean archipelago: the effect of dispersal ability and habitat preference. Molecular ecology, 11, 2503–2517.

Petanidou, T., Ståhls, G., Vujić, A., Olesen, J. M., Rojo, S., Thrasyvoulou, A., et al. (2013). Investigating plant–pollinator relationships in the Aegean: the approaches of the project POL-AEGIS (The pollinators of the Aegean archipelago: diversity and threats). Journal of Apicultural Research, 52(2), 106-117.

Vujic, A., Petanidou, T., Tsheulin, T., Cardoso, P., Radenkovic, S., Stahls, G., et al. (2016). Biogeographical patterns of the genus Merodon Meigen, 1803 (Diptera: Syrphidae) in islands of theeastern Mediterranean and adjacent mainland. Insect Conservation and Diversity.

APPENDIX 1. ILLUSTRATION OF MORPHOLOGICAL STATES *=outgroop state

Body 0 Coloration:

1. Abdomen metallic black or black/orange, 2. All body (excl. front legs) metallic light thorax and legs (2nd,3rd) metallic green green*

Head parts 1 Last maxillary palp segment size ratio to the two segments before:

3. last smaller* 4. +/-same length

5. last bigger

2 Groove at last segment of maxillary palp:

6. wide and deep* 7. thin and deep

8. wide and flat 9. wide and deeply flattened 3 Internal colour of 3 last antennae segments:

10. blackened 11. black*

12. alsmost no black

4 Antennae sensor structure (top of last segment) :

13. two parallel grooves 14. one horseshoe shaped groove * 5 Eyes cavity size:

15. narrow * 16. very narrow

Clypeus 6 With glossy bald surface or area with very sparse punctuation:

17. Yes* 18. No

7 Anterior shape:

19. round* 20. rectangular

21. rounded angle

8 Anterio-lateral edges with strongly elevated peaks:

22. Yes * 23. No

9 Middle keel state:

24. absent 25. present

26. flat 10 Punctuation:

27. sparse 28. dense

29. mixed dense and very sparse*

Pronotum 11 Posterio-lateral with 2 glossy bald/ sparse punctuation surfaces:

30. Yes* 31. No 12 Punctuation:

32. sparsely 33. dense 13 Vermiculation:

34. absent 35 irregular

36 straight

14 Posterio-lateral edges:

37 rounded 38 rounded angle

39 angled

15 Medio-apical edge curved apicaly:

40 Yes * 41 No

16 Color: (states: diverse mix (42-50), purple-burgundy (52), light-green(53))

42 darkgreen 43 green

44 yellowgreen 45 purple

46 petrol 47 blue

48 orange-bronze 49 red-bronze

50 greenish red-bronze 51 purple-burgundy

52 light-green*

17 Glossines:

53 matt 54 glossy

Elytra 18 Apex of elytras strongly remoted :

55 Yes* 56 No

19 Thickness (lateral view) :

57 flat 58 high-thick *

20 Apex edge angle:

59 rounded, no angle* 60 vertical

61 outwards 62 inwards

21 Apex with spike:

63 Yes 64 No

22 Posteriorly black (of females) :

65 Yes 66 No

67 Mixed *

Scutellum 23 Shape:

68 triangular 69 short-rounded *

Hairs 24 Black and white elytral patern:

70 Yes* 71 No

25 Black elytral bristles length:

72 short 73 long

26 White haired body with orange haired abdomen:

74 Yes * 75 No. Body and abdomen hair same color

27 Hair at last tergite (of females) :

76 short 77 long

28 Intense orange hair all over body:

78 Yes 79 No 29 Elytral bristles at apex edge:

80 intense 81 week 30 Black bristles at elytra surface (excl. edge):

82 absent 83 thin

84 thick 85 thin and thick mixed

31 Small lying elytral hair:

86 absent 87 thick

88 thin

32 White-orange elytral bristles :

89 absent 90 present

91 present but different (thin hair, no bristles) *

33 White long and thin elytral hair:

92 present * 93 absent (only as bristles)

34 Whtite elytral bristles olny at the inside edge :

94 Yes * 95 No

35 Black bristles intense all over elytra surface:

96 Yes 97 No

36 Lateral sternite hair:

98. black (orange/white only at top) 99. white-orange

100. mixed white/orange with black 101. mixed small black with white spikes/hairs

102. long thin white-orange*

Legs 37 Mesotibia and metatibia covered with thick spikes :

103. Yes 104. No 38 Apical spurs of metatibia colour:

105. half black half white 106. black with brown tip* 39 Bristels at apical part of protibia colour :

107. black 108. dark brown

109. very light brown

40 Protibia colour:

110. metallic green * 111. metallic dark colors

41 Nails of front leg:

112. almost not curved* 113. lightly curved

114. curved 115. strongly curved

Abdomen 42 Last abdominal segments red-brown (of males) :

116. Yes 117. No*

43 Last abdominal segment big and strongly protrubes downwards (of males) :

118. Yes 119. No*

44 5th sternite very enlarged :

120. No* 121. Yes

45 Sternites V shaped (of females) :

122. Yes 123. No * 46 Last tergite size (of females) :

124. length +/- equal to width 125. length < width

Genitalia 47 Genitalia colour (of males) :

126. black * 127. brown

48 Genitalia types (of males) :

128. Type 1, dorsal (middle wide,thinner to apex) 129. Type 1, lateral

130. Type 2, dorsal view (thnin and tweezer shaped) 131. Type 2, lateral view

132. Type 3, dorsal (paddle shaped) 133. Type 3, lateral

134. Type 4. dorsal (middle and apex same wide) 135. Type 4, lateral

136. Tyoe 5, dorsal (apex wider than middle) 137. Tyoe 5, lateral

138. Type 6, dorsal * 139. Type 6, lateral (round shaped) *

49 Type3 genitalia (paddle shaped) variation discription (males) :

140 normal paddle shaped 141 thinner variation (simillarity with type 2, but inner apex sides aren’t parallel)

50 (Legs) # of toothings at basal part of last protarsus segment (of males):

142. 1 or 2 or 3 or 4 or 5 or 6 or 7

APPENDIX 2. CHARACTER STATE EVALUATION By clicking this link below, a pdf file with the character state evaluation will appear. All the specimens have been examined and were given a value state for each morphological character. In the first column is the under examination body section mentioned, in the second and third is the character number and description, with the description of the possible states in the fourth. The coloring in the first column is also giving information about the characters. In specific, green stands for strong characters, light green for week, blue color is representing the male characters and red the female. After the fifth column and downwards, begins the state evaluation for each specimen, of which the species, gender, island and code information is mentioned in row A,B and C. As also mentioned in Table 2 asterisk stands for outgroup (Eulasia pareyssei) characters and character states. https://docs.google.com/spreadsheets/d/1499PPSKlN6bfQx-vTtigIzSdlqViBra4XwsfUDzs- Uo/pub?gid=155573365&single=true&output=pdf

APPENDIX 3. PHYLOGENETIC TREE For an higher resolution of the phylogenetic tree, as a pdf file, follow the available link: https://drive.google.com/file/d/0Bww89sOaYEypSE10ak9wYi1Sd0U/view?usp=sharing

APPENDIX 4. SPECIES ILLUSTRATION Below are illustrations of the 5 Pygopleurus species. On the right part of the image are all the possible colorations of the sampled specimens.

Pygopleurus foina

male female

Pygopleurus kareli

male female

Pygopleurus labaumei

male female

Pygopleurus vulpes

male female

Pygopleurus medius

male female