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Theses and Dissertations

2020-03-23

Advances in the Systematics and Evolutionary Understanding of Fireflies (Coleoptera: Lampyridae)

Gavin Jon Martin Brigham Young University

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BYU ScholarsArchive Citation Martin, Gavin Jon, "Advances in the Systematics and Evolutionary Understanding of Fireflies (Coleoptera: Lampyridae)" (2020). Theses and Dissertations. 8895. https://scholarsarchive.byu.edu/etd/8895

This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected]. Advances in the Systematics and Evolutionary Understanding of Fireflies

(Coleoptera: Lampyridae)

Gavin Jon Martin

A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Seth M. Bybee, Chair Marc A. Branham Jamie L. Jensen Kathrin F. Stanger-Hall Michael F. Whiting

Department of Biology

Brigham Young University

Copyright © 2020 Gavin Jon Martin

All Rights Reserved

ABSTRACT

Advances in the Systematics and Evolutionary Understanding of Fireflies (Coleoptera: Lampyridae)

Gavin Jon Martin Department of Biology, BYU Doctor of Philosophy

Fireflies are a cosmopolitan group of bioluminescent beetles classified in the family Lampyridae. The first catalogue of Lampyridae was published in 1907 and since that time, the classification and systematics of fireflies have been in flux. Several more recent catalogues and classification schemes have been published, but rarely have they taken phylogenetic history into account. Here I infer the first large scale anchored hybrid enrichment phylogeny for the fireflies and use this phylogeny as a backbone to inform classification. Several classification changes are made throughout the group with emphasis on morphological traits that support the AHE hypothesis.

Building off of this classification work, and in an effort to help correct taxonomic issues that have plagued the Lampyridae, I also present an electronic identification tool to the firefly genera of the world. This tool is built in Lucid and incorporates 23 characters (features) and 76 character states. These characters are inspired by current and historic literature. Emphasis was given to characters and states that are easily located and do not require complex dissection. The key currently works for 113 of the 146 known lampyrid genera. As such, it should be noted that it is a provisionary attempt at identification, and all identifications should be checked against primary literature.

Fireflies, like many organisms, rely on sensory cues from their environment and are an ideal system for studying sensory niche adaptation. This is due in large part to the dependence of many species on bioluminescent sexual communication. Using transcriptomics, I examine the phototransduction pathway and provide some of the first evidence for positive selection in beetles, of components of the phototransduction pathway beyond opsins.

Based on preliminary data gathered in several BYU Bio-100 courses for non-majors, I observed that many students come to class with a human-centric view of the world.. In addition to this, and perhaps as an explanation, students also come to class without a firm understanding of natural history collections and their roles both to the general public and specifically to science. Therefore, in two sections of BIO-100 at BYU students were given an online module as part of their normal homework. This module was designed to use fireflies from the Monte L Bean Science Museum to introduce students to the concept of natural history museums and to give an example of an organism at risk for extinction. Unfortunately, no gain in pro-environmental thinking was observed post-intervention, however, I did observe gains in student’s appreciation of the importance of natural history collections to both the general public and to scientific research.

Keywords: classification, lucid, key, phototransduction, natural history collections

ACKNOWLEDGEMENTS

I would like to thank each member of my committee for the hours of time spent mentoring me not only in science but also in life. I particularly want to thank my committee chair, Dr. Seth Bybee, for the many lessons learned. Dr. Bybee gave me a chance to expand my passion and scope, and I will be forever grateful for the experiences here. I want to thank Dr.

Jamie Jensen especially for helping me develop the tools and skills I will need to be a better educator. I want to thank Dr. Marc Branham for opening his lab and continuing to foster my love for a group of the most fascinating creatures on Earth. I thank Dr. Kathrin Stanger-Hall for pushing me to be a better scientist and a better communicator. I also want to thank Dr. Michael

Whiting for pushing me to always be better, in every aspect of my science.

I want to thank the many curators and collection managers who opened the doors to their collections and without whom this research would not have been possible: Cate Lemann (ANIC),

Shawn Clark (BYU), Chris Grinter (CAS), Paul Skelley and Kyle Schnepp (FSCA), Azadeh

Taghavian (MNHN), Max Barclay and Michael Geiser (NHM), Isabelle Zuercher (NHMB), and

Floyd Shockley (USNM).

I want to also acknowledge my family, who share just as much of this degree as I do. My wife, Tara, has spent countless hours counseling me and reminding me why we do this.

She has sacrificed greatly in this academic pursuit, and through her dedication and love, continually keeps me focused on perspective. I also want to thank my children Emma, Charlie,

Annabelle, and Theodore for their sacrifice and love.

This research was supported financially by grants from the National Science Foundation and the BYU Graduate Mentoring Award.

Table of Contents

Title Page ...... i Abstract ...... ii Acknowledgments ...... iii Table of Contents ...... iv List of Tables ...... v List of Figures ...... vi Chapter 1: Higher-level phylogeny and reclassification of Lampyridae (Coleoptera: Elateroidea) ...... 1 Introduction ...... 3 Materials and Methods ...... 5 Results ...... 10 Discussion ...... 13 Conclusions ...... 22 Acknowledgements ...... 37 Literature Cited ...... 38 Ch. 1 Tables ...... 58 Ch. 1 Figures ...... 63 Chapter 2: Lampyrid-ID: An interactive LUCID based electronic identification key to the genera of the Lampyridae (Coleoptera) of the world ...... 65 Introduction ...... 66 Materials and Methods ...... 67 Conclusions ...... 71 Acknowledgements ...... 73 Literature Cited ...... 74 Ch. 2 Tables ...... 75 Ch. 2 Figures ...... 81 Chapter 3: The genes of the phototransduction pathway among fireflies (Coleoptera: Lampyridae) ...... 82 Introduction ...... 84 Materials and Methods ...... 87 Results ...... 90 Discussion ...... 93 Conclusions ...... 96 Acknowledgements ...... 98 Literature Cited ...... 99 Ch. 3 Figures ...... 108 Chapter 4: Building appreciation for Natural History Collections from biological collection data...... 110 Introduction ...... 111 Materials and Methods ...... 118 Results ...... 121 Discussion ...... 122 Conclusions ...... 124 Acknowledgements ...... 125

iv

Literature Cited ...... 126 Ch. 4 Tables ...... 132 Ch. 4 Figures ...... 133 Appendix ...... 134

v

List of Tables

Chapter 1

Table 1: Comparison of major classification schemes in Lampyridae ...... 58 Table 2: Taxa included in current study ...... 59

Chapter 2

Table 1: Genera and species used for coding of key-matrix ...... 75

Chapter 4

Table 1: Summary statistics of paired differences for paired samples test ...... 132

vi

List of Figures

Chapter 1

Figure 1: Maximum Likelihood reconstruction of full, untrimmed dataset aligned in MAFFT ...... 63 Figure 2: Lamprohizinae habitus images ...... 64

Chapter 2

Figure 1: Ventral habiti of various species showing diversity of photic organ and antennal morphology ...... 81

Chapter 3

Figure 1: Diagram of the basic insect phototransduction pathway (modified from Macias-Munoz et al. 2019) ...... 108 Figure 2: Distributions of the PP of positive selection across phototransduction genes ...... 109

Chapter 4

Figure 1: Scaled bar graph of responses for question 16 ...... 133 Figure 2: Scaled bar graph of responses for question 17 ...... 133

vii

Citation for chapter 1: Martin GJ, Stanger-Hall KF, Branham MA, Da Silveira LF, Lower SE, Hall DW, Li XY, Lemmon AR, Moriarty Lemmon E, Bybee SM. Higher-Level Phylogeny and Reclassification of Lampyridae (Coleoptera: Elateroidea). Insect Systematics and Diversity. 2019 Nov;3(6):ixz024. I hereby confirm that the use of this article is compliant with all publishing agreements.

Chapter 1: Higher-level phylogeny and reclassification of Lampyridae (Coleoptera: Elateroidea)

Gavin J. Martin a*, Kathrin F. Stanger-Hallb, Marc A. Branhamc, Luiz F. L. Da Silveirab, Sarah E.

Lowerd&e, David W. Halld, Xue-Yan Lif, Alan R. Lemmong, Emily Moriarty Lemmonh, Seth M.

Bybee a,i a Department of Biology, 4102 LSB, Brigham Young University, Provo, UT 84602, USA b Department of Plant Biology, University of Georgia, Athens, GA 30602, USA c Department of Entomology & Nematology, University of Florida, Gainesville, FL 32611, USA d Department of Genetics, University of Georgia, Athens, GA 30602, USA e Department of Biology, Bucknell University, Lewisburg, PA 17837, USA f Max-Planck Junior Scientist Group on Evolutionary Genomics, Kunming Institute of Zoology,

Chinese Academy of Sciences, 32 Jiaochang Donglu Kunming, Yunnan 650223 g Department of Scientific Computing, Florida State University, Tallahassee, FL 32306, USA h Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA i Monte L. Bean Museum of Natural History, Brigham Young University, Provo, UT 84602,

USA

* Corresponding author: [email protected]

Keywords: firefly, phylogenomics, target capture, Amydetinae, Lamprohizinae, Pachytarsus,

Crassitarsus, Lamprigera

1 ABSTRACT

Fireflies (Lampyridae: Rafinesque) are a diverse family of beetles which exhibit an array of morphologies including varying antennal and photic organ features. Due in part to their morphological diversity, the classification within the Lampyridae has long been in flux. Here we use an anchored hybrid enrichment approach to reconstruct the most extensive molecular phylogeny of Lampyridae to date (436 loci and 98 taxa) and use this phylogeny to evaluate the higher-level classification of the group. None of the currently recognized subfamilies were recovered as monophyletic with high support. We propose several classification changes supported by both phylogenetic and morphological evidence: (1) Pollaclasis Newman, Vestini

McDermott (incl. Vesta Laporte, Dodacles Olivier, Dryptelytra Laporte, and Ledocas Olivier),

Photoctus McDermott, and Araucariocladus Silveira & Mermudes are transferred to Lampyridae incertae sedis, (2) Psilocladinae Mcdermott, 1964 status novum is reestablished for the genus

Psilocladus Blanchard, (3) Lamprohizini Kazantsev, 2010 is elevated to Lamprohizinae

Kazantsev, 2010 status novum and Phausis LeConte is transferred to Lamprohizinae, (4)

Memoan Silveira and Mermudes is transferred to Amydetinae Olivier, and (5) Scissicauda

McDermott is transferred to Lampyrinae Rafinesque.

2 INTRODUCTION

Fireflies (Lampyridae Rafinesque, 1815) are a cosmopolitan group of beetles with approximately 2200 species (Branham 2010) and a varied and confounded history of classification (Olivier 1907 & 1910a, Green 1959, McDermott 1966, Crowson 1972, Nakane

1991, Jeng 2008). Higher-level firefly classification schemes (e.g. Green 1956) have traditionally been framed in the context of morphological structures related to the production and detection of sexual signals (e.g., photic organs, eyes and antennae). The morphological evolution of these features appears to have been driven by species-specific sexual communication, such as pheromones, glows, and flashes (Buck 1937, Lloyd 1971, Ohba 1983, Branham and Wenzel

2003, Stanger-Hall and Lloyd 2015, Stanger-Hall et al. 2018). Recent work suggests that bioluminescent sexual signals have evolved multiple times during firefly evolution and have been repeatedly lost, with a likely reversal to the use of pheromones for a sexual signal

(Branham and Wenzel 2003, Stanger-Hall et al. 2007, Martin et al. 2017). Therefore, morphologies related to these signals (production/reception) may represent instances of convergent evolution and the continued dependence on these morphologies to support firefly classification and taxonomy (Branham and Wenzel 2003) needs to be reconsidered.

The first catalogue of Lampyridae was authored by Ernst Olivier in 1907 and updated in

1910. In this initial taxonomic effort Olivier included nine subfamilies (based almost entirely on antennal morphology) and approximately 1000 species. Drawing on the earlier work of Green

(1948, 1959), McDermott (1964) rearranged the taxa into seven subfamilies and added a tribal classification for the Lampyrinae. However, he noted that his classification, while logical, did not reflect phylogeny and was “more or less arbitrary,” as it relied heavily on characters such as antennae and photic organ morphology. This work lead to McDermott’s (1966) supplement to

3 the Olivier catalogue. Since McDermott, there have been two revised classification schemes for

Lampyridae (Crowson 1972, Nakane 1991; see Table 1). The classification of the family by

Crowson (1972) was incomplete as it did not include all lampyrid genera, and the work of

Nakane (1991; published in Japanese) has largely been overlooked by current taxonomic workers. Recent phylogenetic efforts have provided deeper insight into the classification of fireflies by expanding morphological data sets (Branham and Wenzel 2001, 2003, Jeng 2008); molecular data sets (Stanger-Hall et al. 2007); or both (Martin et al. 2017). All of these studies highlighted the need to update the higher-level classification of this group within a phylogenetic framework. While focused on the evolution of specific traits, each of these studies recovered non-monophyly of at least one subfamily. We are presenting here the first classification of

Lampyridae that uses extensive taxon sampling and explicitly takes phylogenetic history into account.

4 MATERIALS AND METHODS

Taxon Sampling

Our ingroup sample included 53 (of approximately 145) lampyrid genera and 88 species, representing seven of the eight subfamilies sensu Nakane 1991 (Table 2). However, the

Ototretadrilinae has since been synonymized under the Ototretinae (Janisova and Bocakova

2013). DNA grade material for the Cheguevarinae was not available. The appropriate sister taxon to Lampyridae has been a topic of debate (Branham and Wenzel 2001, 2003, Stanger-Hall et al. 2007, Jeng 2008, Bocakova et al. 2007, Kundrata et al. 2014, Martin et al. 2017), therefore ten outgroup taxa from several closely related elateroid families were sampled: Omethidae,

Eucnemidae, Cantharidae, Lycidae, Elateridae, Rhagophthalmidae, and Phengodidae (Table 2).

DNA Extraction

Specimens were collected in the field, preserved in 95% ethanol and stored at – 80 °C for long-term storage. Each specimen was independently identified by at least two co-authors.

Muscle tissue was removed from a single metacoxae, when possible, for each specimen. In some instances, the small size of the specimen necessitated more of the specimen to be used. DNA was then extracted using a Qiagen DNeasy extraction kit. DNA samples were sent to the Center for

Anchored Phylogenomics at Florida State University, Tallahassee, FL, USA for anchored hybrid enrichment sequencing. The specimens and remaining DNA samples were deposited as vouchers in the BYU Insect Genomics Collection in the Monte L. Bean Museum (BYU) of Natural

History, Provo, UT, USA, the Lampyridae cryo-collection in the Stanger-Hall lab (UGA),

Athens, GA , USA, and the Coleção Entomológica Prof. José Alfredo Pinheiro Dutra,

5 Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

(DZRJ); see table 2.

Probe Design

In collaboration with the Center for Anchored Phylogenomics

(www.anchoredphylogeny.com), an AHE kit representing the diversity of Lampyridae was developed, targeting a subset of the low-copy AHE 941 loci (exons) that Haddad et al. (2018) developed for Coleoptera. To this end, genomic resources, assembled genomes, assembled transcriptomes, and unassembled low-coverage genomic reads (unpublished; see Supp. Table 1 for details), for 14 species of Lampyridae were gathered. In order to collect the low coverage genomic reads, Illumina libraries were prepared following Lemmon et al. (2012) and each was sequenced at 5x coverage on an Illumina HiSeq 2500 with a 150bp paired-end protocol with an

8bp indexing read. After demultiplexing with no mismatches tolerated, overlapping reads were merged following Rokyta et al. (2012).

Target regions appropriate for Lampyridae were identified by following the general methodologies described in Hamilton et al. (2016). The assembled genomes and transcriptomes were scanned using all of the 941 Tribolium castaneum reference sequences developed by

Haddad et al. (2018). After reads were mapped and extended to the same references, the reads were extended up to 2000bp in each direction to produce longer sequences. The resulting sequences were then aligned in Mafft v. 7.407 (Katoh and Standley 2013) and visually inspected in Geneious. Well-aligned regions were selected for downstream analysis. Of the 941 beetle exons identified by Haddad et al. (2018), only 586 regions of >180bp in which at least 16 of 19

6 taxa were present were identified. Seventy overlapping-loci were removed. Repetitive regions were masked using analyses of kmer distributions (see Hamilton et al. 2016 for details).

The final target regions were comprised of 516 target loci covering 578,322 bp. Probes were tiled at 2x coverage across each of the 19 lineages represented in the alignments. After removing redundant probes, 56979 probes were submitted to Agilent Technologies for synthesis

(SureSelect XP kit).

Library Preparation

From extracted DNA, libraries with standard Illiumina adapters were prepared following

Lemmon et al. (2012) and Prum et al. (2015) using a Beckmann Coulter FXp liquid-handling robot. After adding 8bp indexes, libraries were pooled in groups of ~16 and enriched using the

Lampyridae-specific enrichment kit described above. Enriched library pools were then pooled and sequenced on three Illumina 2500 sequencing lanes with C-bot clustering and a paired-end

150bp protocol (~100Gb of raw data were collected).

Read Assembly and Orthology Assessment

Raw reads passing the CASAVA high-chastity filter were merged following Rokyta et al.

(2012) and assembled using the quasi-de novo approach described by Hamilton et al, (2016), with probe region sequences from Phausis reticulata, Pyractomena borealis, Photinus pyralis,

Photinus scintillans, Phausis reticulata and a soldier beetle (Cantharidae), serving as references.

Assembly clusters derived from less than 42 reads were removed from downstream analyses in order to mitigate the effects of any low-level contamination (clusters were typically comprised of

100-1000 reads). Homologous consensus sequences were derived from the remaining clusters,

7 with ambiguous base calls being made for each site with a base pattern that could not be explained by sequencing error, which was assumed to be no more than 1%. For each of the 516 target loci, orthologous sets of homologs were determined using a distance matrix (constructed by comparing kmer distributions) and a neighbor-joining approach for clustering (with at most one sequence per individual allowed in each ortholog cluster). After removing ortholog sets with less than 50% species representation (<51 of 98), 436 loci remained (Supp. Table 2).

Alignment

The sequences obtained for individual loci were aligned via Mafft v. 7.407 (Katoh and

Standley 2013) using the default automatic alignment option. For Maximum Likelihood (ML) analyses, the alignments of the individual loci included flanking regions (head, tail, or both) of each locus and were concatenated before analysis and resulted in an alignment length of

1,833,533 base pairs. Breinholt et al. (2017) showed that trimming data from the probe or flanking region has little effect on the final topology, consequently, we elected not to trim these regions (Lemmon et. al. 2012). However, it is possible that missing data may bias phylogenetic reconstruction (Lemmon et al. 2009; however, see also Wiens and Morrill 2011), therefore, we generated a second dataset utilizing a portion of the pipeline of Breinholt et al. (2017)

(alignment_DE_trim.py) to remove sites from the alignment with <75% density, (i.e. completeness), and with entropy >1.5 (to account for sites estimated to be nearly random). This resulted in a trimmed alignment of 401,423 base pairs.

8 Phylogenetic reconstruction

Maximum Likelihood reconstruction was performed in IQ-Tree v 1.6.8 (Nguyen et al.

2015, Chernomor et al. 2016) with 1,000 ultrafast bootstrap iterations (UFBoot; Hoang et al.

2018). Model estimation for concatenated locus datasets was carried out using ModelFinder

(Kalyaanamoorthy et al. 2017) implemented within IQ-Tree, using BIC criteria. GTR+F+R9 was selected as the best-fit model for the untrimmed dataset and GTR+R9 was selected for the trimmed dataset. To account for the potential effect of incomplete lineage sorting on our phylogenetic reconstruction we also used a coalescent approach to estimate a species tree via

ASTRAL-III (Zhang et al. 2018) for each dataset (trimmed and untrimmed). ML gene tree topologies for each locus were inferred via IQ-Tree (sensu Breinholt et al. 2017) before estimating the ASTRAL topology for each dataset. We used localized posterior probabilities

(Sayyari and Mirarab 2016) to assess nodal support for the coalescent species trees as assessed in

ASTRAL-III.

9 RESULTS

Phylogenetic reconstruction

Maximum Likelihood Reconstruction

Within the maximum likelihood (ML) reconstruction of the untrimmed dataset

(Topology 1 (T1); Fig. 1), we find strong support for a sister group relationship between the

Elateridae, Rhagophthalmidae, and Phengodidae with the Lampyridae (Fig. 1). The Luciolinae +

Lamprigera Motschulsky were recovered as the sister lineage to all other firefly subfamilies with strong support (UFboot 100%). Within the lineage of the remaining subfamilies, a clade comprised of (Pterotus LeConte + Pollaclasis Newman) + the monophyletic Ototretinae forms the sister group to the remaining fireflies. However, neither this clade, nor the Ototretinae clade are well supported (UFBoot: 72% for both nodes). The clade (Lamprohiza Motschulsky +

Phausis LeConte) is recovered as sister to the clade ((Psilocladus Blanchard + (Amydetinae +

Photurinae)) + Lampyrinae) with UFBoot support of 100% at each node.

The ML reconstruction of the trimmed dataset (Topology 2 (T2); Supp. Fig. 1) was largely identical to T1, with the exception of the placement of the paraphyletic Ototretinae and

Pollaclasis in T2. In T2, Ototretinae is rendered paraphyletic, as Stenocladius Fairmaire in

Deyrolle and Fairmaire is recovered as the sister taxon to all other Lampyridae, while Drilaster

Kiesenwetter is recovered as the sister taxon to a clade comprised of Pterotus + Pollaclasis, albeit with low support (UFBoot 77%). Compared to T1, there is also lower support along the backbone of T2 (Supp. Fig. 1).

10 Coalescent Reconstruction

The coalescent reconstruction of the untrimmed dataset (Topology 3 (T3); Supp. Fig. 2) was largely congruent with the ML topology of the same dataset, except for the placement of the

Ototretinae (incl. Pterotus). In the coalescent analysis (T3), the Ototretinae are rendered polyphyletic by inclusion of Pterotus, and this clade is recovered as the sister group to all other fireflies, while in the ML analysis (T1) this position was held by Luciolinae + Lamprigera (Fig.

1; Localized Posterior Probability (LPP): 0.95).

The coalescent reconstruction of the trimmed dataset (Topology 4 (T4); Supp. Fig. 3) was identical in respect to subfamilial relationships in the untrimmed data set (T3), with only minor variation in species relationships within the major clades (i.e. placement of the lucioline clade comprised of Abscondita cerata and Curtos sp.; Supp. Figs. 3 & 4).

Consensus among analyses

In all analyses, Lampyridae was reconstructed as monophyletic with support values of

UFboot: 100% or LPP: 1. With the exception of the Ototretinae (discussed separately below), our four different analyses yielded no topological differences that impact the monophyly of the subfamilies among Lampyridae. Therfore we will use Topology 1 (Fig. 1), to discuss the phylogenetic relationships among the seven subfamilies sampled. Topology 1 (T1) is also the best supported (via ultrafast bootstrap analysis), and is based on the full, untrimmed dataset.

In T1, the Luciolinae are reconstructed as paraphyletic with the inclusion of Lamprigera.

The Ototretinae were represented by two genera (Drilaster and Stenocladius) in our analyses, and these formed a monophyletic clade, albeit with low support (72%). T1 is the only topology to recover the Ototretinae as monophyletic and sister to a clade comprised of Pterotus and

11 Pollaclasis. In both T2 and T4 (Supp Figs. 1 & 3) Stenocladius was recovered as the sister lineage to all other fireflies with low support (UFboot: 77% & LPP: 0.75), while in T3 (Supp.

Fig. 2) a clade consisting of Stenocladius + (Drilaster + Pterotus) is recovered as sister to the remaining fireflies (LPP: 0.95). Given this incongruence, the exact position of the Ototretinae among firefly subfamilies remains uncertain. However, in all phylogenetic reconstructions,

Pterotus is either sister to, or a lineage within, the Ototretinae. The subfamily Amydetinae is recovered as polyphyletic and its taxa can be found closely associated with diverse subfamilies:

Psilocladus is recovered as sister to the Amydetinae + Photurinae, Pollaclasis forms a clade with

Pterotus (Pterotinae), Vesta Laporte is recovered within the photurine clade, and Cladodes

Solier, Ethra Laporte, and Scissicauda McDermott are recovered within the Lampyrinae. The

Photurinae are rendered paraphyletic by inclusion of the old-world Vesta (see Martin et al. 2017).

Lampyrinae is recovered as polyphyletic: Lamprigera is recovered within the Luciolinae, and there is high support (UFboot: 100%) for a monophyletic Lamprohiza + Phausis clade outside the Lampyrinae (see above). In addition, several amydetine taxa (Cladodes, Ethra, Scissicauda) cluster within the remaining Lampyrinae.

As such, in T1, the only subfamily recovered as monophyletic, as currently classified, is the Ototretinae. However, across all other topologies, even the Ototretinae are also rendered non- monophyletic.

12 DISCUSSION

The higher-level classification of Lampyridae is in need of revision. Our molecular analyses confirm that a few morphological characters related to sexual communication, many likely convergent, cannot be central to diagnosing a natural classification of Lampyridae. The use of these characters in subfamilial classification needs to be reassessed in light of a more complete phylogenetic history of the group. Our phylogenetic analyses based on 436 loci and 98 taxa provide a robust framework for a revision of the classification of Lampyridae.

Luciolinae

The Luciolinae are a subfamily of fireflies that have received much attention over the years (for a taxonomic treatment see Ballantyne et al. 2015) and seem to share stable morphological characters such as a reduced number of abdominal ventrites in the males (see discussion of Lamprigera below).

Our analyses largely agree with recent morphological analyses in terms of the major groupings of the Luciolinae (Ballantyne and Lambkin, 2013). Thanks in large part to the effort of

Ballantyne and Lambkin (Ballantyne 1987a, Ballantyne and Lambkin 2009, 2013), there also exists a discussion of the aedeagal sheath, which has provided evidence in support of the current taxonomic framework for these taxa. Many of the morphological characters used in these analyses (Ballantyne and Lambkin 2009, 2013, Ballantyne et al. 2015, 2016) and the phylogenetic patterns they support, correlate well with the phylogenetic hypotheses generated from our molecular data. However, some minor differences between previous phylogenetic work and our overall topology do exist. For example, the Atyphella complex is not recovered as the the most highly derived lineage, as it is in Ballantyne and Lambkin (2013). Our molecular dataset

13 recovers this lineage as more basal compared to the other Luciolinae. In addition, the lampyrine genus Lamprigera is recovered as a lineage within the lucioline clade.

Jeng et al. (2000) remarked that the systematic position of Lamprigera was uncertain and would hopefully be more accurately defined through future phylogenetic investigation. Previous analyses have recovered Lamprigera in various positions within the Lampyridae. In 2006, on the basis of the 16S mitochondrial marker, Li et al performed a phylogenetic reconstruction of the

Lampyridae. In this analysis, Stenocladius was recovered as the basal lineage, while Lamprigera was recovered in a surprising clade together with the amydetine genus Vesta sister to the remaining fireflies (Li et al. 2006), albeit with low support. In his thesis, Jeng (2008) recovered

Lamprigera sister to the Phausis + Lamprohiza using >400 morphological characters. Wang et al. (2017) recovered Lamprigera as a member of the Lampyrinae on the basis of 13 mitochondrial genes. It should be noted, however, that the Wang et al. (2017) analysis suffered from a limited taxon sampling (six lucioline taxa and two additional lampyrine taxa from a single genus). Martin et al. (2017) recovered Lamprigera as sister to the monophyletic Luciolinae with high support and placed it as Lampyridae incertae sedis.

Here we recover Lamprigera as a member of the Luciolinae for the first time with both strong support and congruence between all of our analyses. However, this placement is based on a single Lamprigera species and major morphological differences between Lamprigera and the luciolines, e.g. the number of abdominal ventrites, need to be addressed. A major, long-standing morphological synapomorphy for Luciolinae has been males with six abdominal ventrites, whereas all other Lampyridae have seven or eight abdominal ventrites. Males of Lamprigera exhibit the “typical” abdominal morphology of Lampyridae with seven ventrites.

14 In certain elateroid lineages, ventrite number is known to vary greatly, even within recently derived tribes/subfamilies (Kundrata & Bocak 2019). In contrast, ventrite number has not been shown to vary in Luciolinae. To rigorously test the classification of Lamprigera relative to the Luciolinae, an expanded taxon sampling including deeper species coverage within the

Lamrigera, combined with an in-depth morphological investigation, including the plasticity of ventrite number across these taxa will be needed. Until these analyses can be done, we elect to keep Lamprigera as Lampyridae incertae sedis (Martin et al., 2017).

Pterotinae, Cyphonocerinae, and Psilocladinae (McDermott, 1964) stat. nov.

Jeng et al. 1998 & 2006, summarizing the work of McDermott (1966), Crowson (1972), and Nakane (1991), was the first to formally delineate the Psilocladinae (previously known as

Cyphonocerinae), by identifying the constituents of the group (Cyphonocerus Kiesenwetter,

Psilocladus, and Pollaclasis) and laying out nine morphological features uniting the group. Jeng et al. (1998) also recognized Cyphonocerinae as a subjective synonym of Psilocladinae based on priority. In 2016, Silveira et al. treated Scissicauda as a member of the amydetine subtribe

Psilocladina sensu McDermott 1964, distinguishing Scissicauda from the other members of the group (Ethra, Photoctus McDermott, Psilocladus, and Pollaclasis). In 2017, another genus

(Araucariocladus Silveira & Mermudes) was added to the Psilocladina sensu McDermott. As a consequence of recognizing Psilocladina sensu McDermott instead of Psilocladinae sensu

Jeng/Nakane, Cyphonocerus was left as the sole member of the Cyphonocerinae.

Our phylogenetic analyses support in part the classification presented by Crowson (1972) and Nakane (1991) and supports the classification of Psilocladus as a separate lineage from the

Amydetinae. Without Cyphonocerus in our taxon sample, the placement of Psilocladus within

15 the Cyphonocerinae cannot truly be tested, however, given that Psilocladus did not form a monophyletic lineage with Pollaclasis, we formally recognize the subfamily Psilocladinae, distinguished by the antennae with 11 articles, articles 2–11 with two weak, ciliate branches, with the only constituent genus being Psilocladus.

Following McDermott (1966) and Silveira et al. (2016), Pollaclasis is classified as a member of the Amydetinae. However, both our ML and coalescent analyses challenge this classification. The ML analysis places Pollaclasis as a sister taxon to the North American

Pterotus, the sole member of the Pterotinae (Fig. 1). In contrast, the coalescent analysis places

Pollaclasis as sister to a Phausis + Lamprohiza clade (Supp. Fig. 2). Hypothesized to be a close relative of Cyphonocerus, Pollaclasis has previously been classified in the Psilocladinae based on the morphology of antennae, mandibles, and abdominal segmentation (see Jeng et al. 1998).

The present analyses do not support the monophyly of Pollaclasis and Psilocladus. Based on this evidence, as well as the absence of Cyphonocerus in our taxon sample, we transfer Pollaclasis to

Lampyridae incertae sedis. Future efforts need to be made to ascertain whether Pollaclasis is indeed a member of the Cyphonocerinae, or as the ML analyses suggest, more closely related to

Pterotus.

Ototretinae

Our analyses indicate that there may be a close relation between the Ototretinae and the

Pterotinae. Recently, the Ototretinae were revised, and their taxonomic placement was addressed

(Janisova and Bocakova 2013). In that study, the monotypic Ototretadrilinae were synonomized under the Ototretinae, and the ototretine lineage was transferred from Elateriformia incertae sedis to Lampyridae. In addition, the genus Stenocladius was transferred from Elateriformia

16 incertae sedis to Ototretinae. Our analyses corroborate these transfers, however, as our taxon sample included only two of eighteen genera from the Ototretinae, Stenocladius and Drilaster, a wider generic sampling is needed to address the phylogenetic history of this lineage.

Two other genera, Anadrilus Kirsch, 1875 and Pachytarsus Motschulsky, 1861, were both excluded from the Ototretinae by Janisova and Bocakova (2013) on the basis of lack of type material to examine. As we were also unable to sample these genera, they remain Lampyridae incertae sedis. Of note, when he described Pachytarsus, Motschulsky (1861) was apparently unaware of the true bug genus by the same name, having been described the year before:

Pachytarsus Fieber, 1860 (for current usage see Ballal et al. 2018). Due to the name Pachytarsus being preoccupied, we propose the replacement name Crassitarsus Martin.

Crassitarsus Martin

NEW REPLACEMENT NAME

Pachytarsus Motschulsky, 1861

Included species: Crassitarsus basalis (Motschulsky, 1861) NEW COMBINATION,

Crassitarsus bicolor (Pic, 1929) NEW COMBINATION, Crassitarsus bryanti (Wittmer, 1940)

NEW COMBINATION, Crassitarsus lateralis (Motschulsky, 1861) NEW COMBINATION,

Crassitarsus longicornis (Pic, 1921) NEW COMBINATION, Crassitarsus minutus (Pic, 1933)

NEW COMBINATION, Crassitarsus obscurus (Pic, 1927) NEW COMBINATION,

Crassitarsus testaceus (Motschulsky, 1861) NEW COMBINATION.

Etymology: Crassus, a synonym for pachy, both meaning thick, seems an apt replacement to conserve the original thoughts of Motschulsky.

17 Lamprohizinae Kazantsev, 2010 stat. nov.

There is strong support for a Lamprohiza + Phausis clade in all our topologies. This is supported by a high degree of morphological similarity between these two genera (Fig. 2).

Phausis was erected for Lampyris reticulata Say by LeConte (1852) and Lamprohiza was erected in 1853 by Motschulsky for Lampyris splendidula Linnaeus. Lamprohiza was later synonymized with Phausis by Lacordaire in 1857. In contrast, Mulsant, 1862 treated Lamprohiza as an independent genus, but in 1881 LeConte wrote “[Phausis] is not sufficiently distinct from the European Lamprohiza, and in fact the European species seems to have been naturalized in

Maryland and Illinois” and included L. splendidula within Phausis. This classification was accepted until 1964 when McDermott separated Lamprohiza from Phausis by the “minute appendage on the 11th antennal article” of the latter. However, Fender (1966) treated the two genera as one in his treatment on the “Phausis” of North America, while Miksic (1969) followed

McDermott in treating them as separate genera. From a phylogenetic perspective, Stanger-Hall et al. (2007) found Phausis as sister to Photurinae + Lampyrinae, similar to our results. In 2008,

Jeng found support for these genera as members of Lampyrinae, however, he noted that they differed from the traditional Lampyrinae in the “unmodified mandibles sensu Green (1949), dorsal abdominal spiracles, and a symmetrical aedeagal sheath.” Our analyses, based on the type species of each genus, strongly support the rank of subfamily and we herein elevate

Lamprohizini Kazantsev, 2010 to Lamprohizinae Kazantsev, 2010 stat. nov.

Diagnosis

The Lamprohizinae are distinguished from all other subfamilies with the following combination of characters, based on adult males: mandibles unmodified (i.e. not reduced in size);

18 antennae filiform, 11-segmented, with or without terminal sensorium, if without then posterior margin of ventrite 7 with weak to strong medial projection, projection emarginate at midline; tarsal claws simple, not bifid; abdomen with seven–eight ventrites; abdominal spiracles dorsal; aedeagal sheath symmetrical.

Key to the genera of Lamprohizinae

Terminal antennomere with small, beadlike sensorium; abdominal ventrite 7 unmodified; ventrite 8 apparent ……………………….………………………………………………...Phausis

Terminal antennomere without small, beadlike sensorium; abdominal ventrite 7 with weak to strong medial projection; ventrite 8 not apparent ………………………….……….…Lamprohiza

Amydetinae

McDermott (1964) recognized the Amydetini as a tribe within the Lampyrinae based on the mandibles being “normal, arcuate, regularly narrowing to tips,” antennae bearing rami or branches, and lack of secondary elytral pubescence of the males. To further classify the rather heterogenous group of genera included in the Amydetini, McDermott (1964) recognized three subtribes based on variation in antennal morphology: Amydetina (“Antennae with more than 14 articles, uniramose”), Vestina (“Antennae with 11 articles, Antennae uniramose, rami remiform, relatively broad, folding like a fan”), and Psilocladina (“Antennae with 11 articles, Antennae bi- or uniramose, rami either long and diffuse, or if short and straight, not fan-folding"). In his 1966 update to the classification, McDermott elevated the Amydetini to the subfamily level. Nakane

(1991) reduced the Amydetinae to Amydetes Hoffmannsegg in Illiger and Magnoculus

McDermott, however Silveira and Mermudes (2013) described the new genus Memoan Silveira

19 and Mermudes and, based on it having features of both the Amydetinae and the Lampyrinae, placed it in Lampyridae incertae sedis.

Due to the phylogenetic position of Memoan as sister to Amydetes, and the morphological features shared by Memoan with other taxa within the Amydetinae (as identified by Silveira and

Mermudes, 2013), including a “continuous glow, pleuralventral suture, ventral approximate eyes, deep punctures on pronotum and scutellum, and absence of tibial spurs,” we formally transfer

Memoan to the Amydetinae, thus the Amydetinae now include Amydetes, Magnoculus, and

Memoan.

Cheguevariinae

Kazantsev (2006) described two enigmatic species in the genus Cheguevaria Kazantsev,

2006, one from Puerto Rico, and one from Dominican Republic. In the description, he remarks on the similarity of Cheguevaria to the Phengodidae, however, he does note the differences, and allies Cheguevaria with the Lampyridae based on the structure of the genital capsule. While he suspected that the genus represented a unique lampyrid subfamily, given the peculiar morphology of the genus, as well as the lack of phylogenetic stability at the time, Kazantsev placed the Cheguevariini as Lampyridae incertae sedis (Kazantsev, 2006). Recent phylogenetic analyses have corroborated the placement of Cheguevaria within the Lampyridae and, confirming the suspicion of Kazantsev, elevated the tribe to subfamilial status (Ferreira, et al.

2019). We were unable to sample the Cheguevariinae for the present analyses.

20 Photurinae and Lampyrinae

All photurine genera in our analyses (we were unable to sample Presbyolampis Buck,

1947) were recovered in a monophyletic lineage with the old-world Vesta. Known only from the

New World, the Photurinae are typically characterized by bifurcate claws in males, and eyes expanding beyond the hypomeron. Nakane (1991) transferred the members of the Vestina (Vesta,

Cladodes, Ledocas Olivier, Dodacles Olivier, and Dryptelytra Laporte) to the Lampyrinae, and moved Psilocladus to the Cyphonocerinae (see above) and Ethra to the Lampyrinae; he did not mention Scissicauda or Photoctus. Our analyses agree with Nakane (1991) in the placement of

Cladodes and Ethra, however, the old-world Vesta sampled for our analyses formed a monophyletic lineage within the Photurinae, a new-world clade. Vesta is a speciose genus with representatives in both the Old and New World. McDermott (1964) recognized that Vesta might not be a natural group. In his 1966 catalogue he continued to treat the new-world taxa as potentially independent from the old-world taxa. As we were unable to sample the new-world

Vesta, or the other members of the Vestini, our analyses are inconclusive with regard to the subfamilial placement of the Vesta. Therefore, we transfer Vesta and the other members of the

Vestini (Dodacles, Dryptelytra, and Ledocas) to Lampyridae incertae sedis pending further morphological and phylogenetic analyses.

The Lampyrinae are a diverse assemblage of genera lacking any stable morphological characters (McDermott 1964; Jeng 2008). It is in part due to this that we believe so many

“lampyrine” genera are/have been mis-classified. Based on our present phylogenetic analyses, we transfer the genus Scissicauda from Amydetinae into Lampyrinae. Since neither Photoctus nor Araucariocladus were included in the present analyses we transfer these to Lampyridae incertae sedis, pending further phylogenetic analyses.

21 CONCLUSIONS

With 88 lampyrid taxa in 53 genera for seven of the eight previously recognized subfamilies, these analyses represent the largest molecular phylogeny of the group, thereby providing a unique opportunity to robustly test the monophyly of each subfamily. The results of our four phylogenetic reconstructions were largely congruent, with the only major difference concerning the placement of the Ototretinae, which were under-sampled in this study. The inclusion of additional ototretine taxa will likely aid the resolution of their placement. Aside from the monotypic Pterotinae, among the subfamilies in the current analyses, only the

Ototretinae (as currently defined) were reconstructed as monophyletic (T1). As a result, we propose several changes to the higher-level classification of Lampyridae, with an updated higher-level classification scheme for the family. Pollaclasis, Vesta (as well as the other constituents of the Vestini: Dodacles, Dryptelytra, and, Ledocas), Photoctus, and

Araucariocladus are transferred to Lampyridae incertae sedis. Psilocladinae is re-established for

Psilocladus. Amydetinae is limited to Amydetes, Magnoculus, and Memoan with Scissicauda being transferred to the Lampyrinae. Lamprohizini is elevated to the rank of subfamily, Phausis is transferred to Lamprohizinae. This comprehensive classification of firefly lineages will provide the phylogenetic scaffold in support of future studies into the evolution of the fascinating morphology and behavior of fireflies.

List of Genera

Subfamily Luciolinae Lacordaire, 1857

Abscondita Ballantyne, Lambkin, & Fu in Ballantyne et al., 2013

Luciola terminalis Olivier, 1883: 330

22 Aquatica Fu, Ballantyne, and Lambkin, 2010

Aquatica wuhana Fu, Ballantyne, and Lambkin, 2010: 8

Aquilonia Ballantyne in Ballantyne and Lambkin, 2009

Luciola costata Lea, 1921: 66

Asymmetricata Ballantyne in Ballantyne and Lambkin, 2009

Luciola circumdata Motschulsky, 1854b: 50

Atyphella Olliff, 1890

Atyphella lychnus Olliff, 1890: 647

Australoluciola Ballantyne in Ballantyne and Lambkin, 2013

Lampyris australis Fabricius, 1775: 201

Colophotia Motschulsky, 1853

Lampyris praeusta Eschscholtz, 1822: 57

Convexa Ballantyne in Ballantyne and Lambkin, 2009

Luciola wolfi Olivier, 1910b: 343

Curtos Motschulsky, 1845

Curtos mongolicus Motschulsky, 1845: 36

Emarginata Ballantyne in Ballantyne et al., 2019

Luciola trilucida Jeng and Lai in Jeng et al., 2003: 248

Emeia Fu, Ballantyne and Lambkin, 2012

Curtos pseudoauteri Geisthardt, 2004: 1

Inflata Boontop in Ballantyne et al., 2015

Luciola indica Motschulsky, 1854b: 53

Kuantana Ballantyne in Ballantyne et al., 2019

23 Kuantana menayah Ballantyne in Ballantyne et al., 2019: 82

Lampyroidea Costa 1875

Lampyroidea syriaca Costa 1875: CLXIX

Lloydiella Ballantyne in Ballantyne and Lambkin, 2009

Luciola majuscula Lea, 1915: 495

Luciola Laporte, 1833

Luciola pedemontana Motschulsky, 1853: 53

Magnalata Ballantyne in Ballantyne and Lambkin, 2009

Luciola limbata Blanchard, 1853: 73

Medeopteryx Ballantyne in Ballantyne and Lambkin, 2013

Pteroptyx effulgens Ballantyne, 1987b: 141

Missimia Ballantyne in Ballantyne and Lambkin, 2009

Missimia flavida Ballantyne in Ballantyne and Lambkin, 2009

Pacifica Ballantyne in Ballantyne and Lambkin, 2013

Atyphella salomonis Olivier, 1911c: 172

Photuroluciola Pic, 1931

Photuroluciola deplanata Pic, 1931: 12

Pteroptyx Olivier, 1902

Luciola testacea Motschulsky, 1854b: 48

Pygatyphella Ballantyne, 1968

Atyphella obsoleta Olivier, 1911c: 174

Pygoluciola Wittmer, 1939

Pygoluciola stylifer Wittmer, 1939: 22

24 Pyrophanes Olivier, 1885

Pyrophanes similis Olivier, 1885: 370

Sclerotia Ballantyne in Ballantyne et al., 2016

Luciola aquatilis Thancharoen in Thancharoen et al., 2007: 56

Serratia Ballantyne in Ballantyne et al. 2019

Serratia sibuyania Ballantyne in Ballantyne et al., 2019: 147

Triangulara Pimpasalee in Ballantyne et al., 2016

Triangulara frontoflava Pimpasalee in Ballantyne et al., 2016: 242

Trisinuata Ballantyne in Ballantyne and Lambkin, 2013

Trisinuata caudabifurca Ballantyne in Ballantyne and Lambkin, 2013:

117

Tribe Pristolycini Kazantsev, 2010

Pristolycus Gorham, 1883

Pristolycus sagulatus Gorham, 1883: 407

Subfamily Pterotinae LeConte, 1861

Pterotus LeConte, 1859

Pterotus obscuripennis LeConte, 1859: 86

Subfamily Ototretinae McDermott, 1964

Baolacus Pic, 1915

Baolacus lajoyei Pic, 1915: 21

Brachylampis Van Dyke, 1939

Brachylampis sanguinicollis Van Dyke, 1939: 16

Brachypterodrilus Pic, 1918

25 Brachypterodrilus pallidipes Pic, 1918: 1

Ceylanidrilus Pic, 1911

Ceylanidrilus bipartitus Pic, 1911a: 187

Drilaster Kiesenwetter, 1879

Drilaster axillaris Kiesenwetter, 1879: 311

Emasia Bocáková and Janišová, 2010

Emasia dentata Bocáková and Janišová, 2010: 61

Eugeusis Westwood, 1853

Eugeusis palpator Westwood, 1853: 239

Falsophaeopterus Pic, 1911

Falsophaeopterus fruhstorferi Pic, 1911a: 187

Flabellopalpodes Bocakova and Bocak, 2016

Flabellopalpodes flavus Bocakova and Bocak, 2016: 372

Flabellototreta Pic, 1911

Flabellototreta fruhstorferi Pic, 1911b: 156

Gorhamia Pic, 1911

Gorhamia compressicornis Pic, 1911a: 187

Harmatelia Walker, 1858

Harmatelia bilinea Walker, 1858: 281

Hydaspoides Janišová and Bocáková, 2013

Hydaspoides kanarensis Janisova and Bocakova, 2013: 7

Hyperstoma Wittmer, 1979

Hyperstoma marginata Wittmer, 1979: 86

26 Lamellipalpodes Maulik, 1921

Lamellipalpodes annandalei Maulik, 1921: 584

Lamellipalpus Maulik, 1921

Eugeusis nigripennis Pascoe, 1887: 10

Ototretadrilus Pic, 1921

Ototretadrilus atritarsis Pic, 1921: 13

Picodrilus Wittmer, 1938

Ototreta drescheri Pic, 1937: 138

Stenocladius Fairmaire in Deyrolle and Fairmaire, 1878

Stenocladius davidis Fairmaire in Deyrolle and Fairmaire, 1878: 113

Subfamily Lamprohizinae Kazantsev, 2010

Phausis LeConte, 1852

Lampyris reticulata Say, 1825: 163

Lamprohiza Motschulsky, 1853

Lampyris splendidula Linnaeus, 1767: 644

Subfamily Cyphonocerinae Crowson, 1972

Cyphonocerus Kiesenwetter, 1879

Cyphonocerus ruficollis Kiesenwetter, 1879: 312

Subfamily Psilocladinae McDermott, 1964

Psilocladus Blanchard, 1846

Psilocladus miltoderus Blanchard, 1846: 122

Subfamily Amydetinae Olivier in Wytsman, 1907

Tribe Amydetini Olivier in Wytsman, 1907

27 Amydetes Hoffmannsegg in Illiger, 1807

Homalisus apicalis Germar, 1824: 67

Magnoculus McDermott, 1964

Megalophthalmus bennetti Gray, 1832: 371

Memoan Silveira and Mermudes, 2013

Memoan ciceroi Silveira and Mermudes, 2013: 84

Subfamily Cheguevariinae Kazantsev, 2006

Tribe Cheguevariini Kazantsev, 2006

Cheguevaria Kazantsev, 2006

Cheguevaria taino Kazantsev, 2006: 370

Subfamily Photurinae Lacordaire, 1857

Bicellonycha Motschulsky, 1853

Bicellonycha deleta Motschulsky, 1854b: 58

Photuris Dejean, 1833

Lampyris versicolor Fabricius, 1798: 123

Presbyolampis Buck, 1947

Presbyolampis immigrans Buck, 1947: 75

Pyrogaster Motschulsky, 1853

Pyrogaster grylloides Motschulsky, 1853: 53

Subfamily Lampyrinae Rafinesque, 1815

Tribe Cratomorphini Green, 1948

Aspisoma Laporte, 1833

Lampyris ignita Linnaeus, 1767: 645

28 Aspisomoides Zaragoza-Caballero, 1995

Aspidosma bilineatum Gorham, 1880b: 86

Cassidomorphus Motschulsky, 1853

Cassidomorphus silphoides Motschulsky, 1853: 36

Cratomorphus Motschulsky, 1853

Photinus fabricii Laporte, 1840: 268

Micronaspis Green, 1948

Micronaspis floridana Green, 1948: 63

Paracratomorphus Zaragoza-Caballero, 2013

Paracratomorphus reyesi Zaragoza-Caballero, 2013: 145

Pyractomena Melsheimer, 1846

Pyractomena lucifera Melsheimer, 1846: 304

Tribe Lamprocerini Olivier, 1907

Alecton Laporte, 1833

Alecton discoidalis Laporte, 1833: 135

Lamprocera Laporte, 1833

Homalisus grandis Sturm, 1826: 58

Lucernuta Laporte,1833

Lampyris fenestrata Germar, 1824: 66

Lucio Laporte, 1833

Lucio abdominale Laporte, 1833: 136

Lychnacris Motschulsky, 1853

Lychnacris triguttula Motschulsky, 1853: 33

29 Tenaspis LeConte, 1881

Hyas angularis Gorham, 1880: 7

Tribe Lampyrini Rafinesque, 1815

Afrodiaphanes Geisthardt, 2007

Lampyris marginipennis Boheman, 1851: 439

Diaphanes Motschulsky, 1853

Lampyris luniger Motschulsky, 1853: 45

Lampyris Geoffroy, 1762

Lampyris noctiluca Linnaeus, 1767: 643

Lychnobius Geisthardt, 1983

Lampyris conspicua Gyllenhal in Schönherr, 1817: 20

Microlampyris Pic, 1956

Microlampyris basilewski Pic, 1956: 193

Microphotus LeConte, 1866

Microphotus dilatatus LeConte, 1866: 90

Nelsonphotus Cicero, 2006

Nelsonphotus aridus Cicero, 2006: 201

Nyctophila Olivier, 1884

Lamprotomus caucasica Motschulsky, 1854a: 19

Ovalampis Fairmaire, 1898

Ovalampis crispaticollis Fairmaire, 1898: 404

Paraphausis Green, 1949

Paraphausis eximia Green, 1949: 4

30 Pelania Mulsant, 1860

Lampyris mauritanica Linnaeus, 1767: 645

Petalacmis Olivier, 1908

Petalacmis praeclarus Olivier, 1908: 187

Prolutacea Cicero, 2006

Lampyris pulsator Cicero, 1984: 322

Pyrocoelia Gorham, 1880

Lampyris bicolor Fabricius, 1801: 100

Tribe Photinini LeConte, 1881

Ankonophallus Zaragoza-Caballero and Navarrete-Heredia, 2014

Ankonophallus zuninoi Zaragoza-Caballero and Navarrete-Heredia, 2014:

126

Aorphallus Zaragoza-Caballero and Gutierrez-Carranza, 2018

Aorphallus cibriani Zaragoza-Caballero and Gutierrez-Carranza, 2018:

160

Callopisma Motschulsky, 1853

Lampyris rufa G.A. Olivier, 1790: 28

Calotrechelum Pic, 1930

Calotrachelum olivieri Pic, 1930: 88

Dadophora Olivier in Wytsman, 1907

Dadophora hyalina Olivier in Wytsman, 1907: 27

Dilychnia Motschulsky, 1853

Dilychnia basalis Motschulsky, 1853: 30

31 Ellychnia Blanchard, 1845

Lampyris corrusca Linnaeus, 1767: 644

Erythrolychnia Motschulsky, 1853

Erythrolychnia dimidiatipennis Motschulsky, 1853: 29

Heterophotinus Olivier, 1894

Photinus limbipennis Du Val, 1857: 86

Jamphotus Barber, 1941

Jamphotus tuberculatus Barber, 1941: 11

Lucidina Gorham, 1883

Lucidina accensa Gorham, 1883: 408

Lucidota Laporte, 1833

Lucidota banoni Laporte, 1833: 137

Lucidotopsis McDermott, 1960

Lucidota cruenticollis Fairmaire, 1889: 38

Luciuranus Silveira, Khattar, and Mermudes in Silveira et al., 2016

Luciuranus josephi Silveira, Khattar, and Mermudes in Silveira et al.,

2016 377

Macrolampis Motschulsky, 1853

Macrolampis longipennis Motschulsky, 1853: 37

Microdiphot Barber, 1941

Microdiphot cavernarum Barber, 1941: 13

Mimophotinus Pic, 1935

Mimophotinus angustatus Pic, 1935: 9

32 Oliviereus Pic, 1930

Oliviereus flavus Pic, 1930: 88

Phosphaenopterus Schaufuss, 1870

Phosphaenopterus metzneri Schaufuss, 1870: 61

Phosphaenus Laporte, 1833

Lampyris hemiptera Geoffroy in Fourcroy, 1785: 58

Photinoides McDermott, 1963

Photinoides penai McDermott, 1963: 87

Photinus Laporte, 1833

Lampyris pallens Fabricius, 1798: 124

Platylampis Motschulsky, 1853

Platylampis latiuscula Motschulsky, 1853: 44

Pseudolychnuris Motschulsky, 1853

Pseudolychnuris vittata Motschulsky, 1853: 32

Pyractonema Solier in Gay, 1849

Pyractonema compressicornis Solier in Gay, 1849: 446

Pyropyga Motschulsky, 1852

Lampyris nigricans Say, 1823: 179

Pyropygodes Zaragoza-Caballero, 2000

Pyropygodes huautlae Zaragoza-Caballero, 2000: 20

Robopus Motschulsky, 1853

Robopus roseicollis Motschulsky, 1853: 42

Rufolychnia Kazantsev, 2006

33 Callopisma borencona Leng and Mutchler, 1922: 440

Uanauna Campello-Gonçalves, Souto, Mermudes, and Silviera, 2019

Uanauna angaporan Campello-Gonçalves, Souto, Mermudes, and Silviera,

2019: 67

Ybytyramoan Silveira and Mermudes, 2014

Ybytyramoan praeclarum Silveira and Mermudes, 2014: 328

Tribe Pleotomini Summers, 1875

Calyptocephalus Gray in Griffith and Pidgeon, 1832

Calyptocephalus fasciatus Gray in Griffith and Pidgeon, 1832: 70

Ophoelis Olivier, 1911

Ophoelis impura Olivier, 1911: 48

Phaenolis Gorham, 1880

Phaenolis laciniatus Gorham, 1880: 10

Pleotomodes Green, 1948

Pleotomodes needhami Green, 1948: 65

Pleotomus LeConte, 1861

Pleotomus pallens LeConte, 1866: 88

Roleta McDermott, 1962

Roleta coracina McDermott, 1962: 69

Lampyrinae incertae sedis

Cladodes Solier in Gay, 1849

Cladodes flabellatus Solier in Gay, 1849: 445

Ethra Laporte, 1833

34 Cladophorus marginatus Gray in Griffith and Pidgeon, 1832: 371

Scissicauda McDermott, 1964

Lucidota disjuncta Olivier, 1896: 1

Lampyridae incertae sedis

Anadrilus Kirsch, 1875

Anadrilus indus Kirsch, 1875: 37

Araucariocladus Silveira and Mermudes, 2017

Araucariocladus hiems Silveira and Mermudes, 2017: 209

Crassitarsus Martin, nom. nov.

Pachytarsus basalis Motschulsky, 1861: 135

Lamprigera Motschulsky, 1853

Lamprigera boyei Motschulsky, 1853: 48

Oculogryphus Jeng, Engel, and Yang, 2007

Oculogryphus fulvus Jeng in Jeng, Engel, and Yang, 2007: 7

Photoctus McDermott, 1961

Photoctus boliviae McDermott, 1961: 174

Pollaclasis Newman, 1838

Pollaclasis ovatus Newman, 1838: 385

Subtribe Vestini McDermott, 1964

Dodacles Olivier, 1885

Dodacles elegans Olivier, 1885: 141

Dryptelytra Laporte, 1833

Dryptelytra cayennenis Laporte, 1833: 139

35 Ledocas Olivier, 1885

Ledocas parallelus Olivier, 1885: 140

Vesta Laporte, 1833

Vesta chevrolati Laporte, 1833:133

36 ACKNOWLEDGMENTS

We would like to thank the members of the Bybee and Whiting labs at Brigham Young

University, the Stanger-Hall lab at the University of Georgia, and the Branham lab at the

University of Florida for specimen acquisition. We also specifically thank Brian Cassell, Joe

Cicero, Raphael DeCock, E. Evans, Lynn Faust, Jim Lloyd, Arwin Provonsha, P. Smith, Fred

Vencl, and Ming-Luen Jeng for collecting specimens (Stanger-Hall lab collection). We thank

Michelle Kortyna, Kirby Birch, and Sean Holland at the Center for Anchored Phylogenomics for assistance with data collection and analyses. We also thank Jesse Breinholt, Robert Erickson,

Anton Suvorov, and Kyle Warren for assistance and advice on analyses, as well as the Fulton

Supercomputing Group at BYU where all analyses were performed. We want to thank Michael

Ivie, Oliver Keller, Nathan Lord, Yelena Pacheco, Gareth Powell, and Caroline Storer for general comments on the manuscript. We thank Floyd Shockley (USNM), Azadeh Taghavian

(MNHN), Isabelle Zuercher (MNB), and Max Barclay and Michael Geiser (NHM) for access to museums for specimen examination and for imaging of Phausis reticulata (USNM) and

Lamprohiza delarouzei (MNHN). This project was funded via the BYU MEG awarded to SMB and NSF: DEB-1655981 (SMB), DEB-1655908 (KSH) and DEB-1655936 (MAB).

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57 CHAPTER 1 TABLES

Table 1. Comparison of major classification schemes in Lampyridae. Olivier 1910 McDermott 1966 Crowson 1972 Nakane 1991 Amydetinae Amydetinae Amydetinae Amydetinae Luciolinae Luciolinae Luciolinae Luciolinae Photurinae Photurinae Photurinae Photurinae Lampyrinae Lampyrinae Lampyrinae Lampyrinae Dadophorinae Lamprocerinae Photininae Lucidotinae Megalophthalminae Pterotinae Pterotinae Pterotinae Matheteinae Rhagophthalminae Ototretinae Ototretinae Cyphonocerinae Psilocladinae Ototretadrilinae Ototretadrilinae

58 Table 2. Taxa included in current study. Coll. Code Family Subfamily Species CO1548 Cantharidae Cantharidae sp. 1 BYU KSH_12271 Cantharidae Cantharidae sp. 2 UGA

CO1310 Elateridae Ampedus pomonae BYU

CO1077 Elateridae Pyrophorus sp. BYU

CO1309 Eucnemidae Microrhagus pygmaeus BYU

CO1545 Lycidae Calopteron reticulatum BYU

CO1286 Omethidae Telegeusis nubifer BYU

CO1544 Phengodidae Phengodes sp. BYU

CO1285 Phengodidae Zarhipis integripennis BYU CO1304 Rhagophthalmidae Rhagophthalmus ohbai BYU

CO1212 Lampyridae Amydetinae Amydetes fastigiata DZRJ

CO1211 Lampyridae Amydetinae Cladodes illigeri DZRJ CO1213 Lampyridae Amydetinae Ethra axillaris DZRJ

CO1214 Lampyridae Amydetinae Memoan ciceroi DZRJ

CO1216 Lampyridae Amydetinae Psilocladus sigillatus DZRJ

CO1245 Lampyridae Amydetinae Psilocladus sp. 1 BYU

CO1218 Lampyridae Amydetinae Psilocladus sp. 2 BYU

CO1215 Lampyridae Amydetinae Scissicauda disjuncta DZRJ

CO1305 Lampyridae Amydetinae Vesta impressicollis BYU

CO1299 Lampyridae Amydetinae Vesta saturnalis BYU

CO1219 Lampyridae Amydetinae Vesta sp. BYU

CO1281 Lampyridae Cyphonocerinae Pollaclasis bifaria BYU

CO1069 Lampyridae Lampyrinae Aspisoma sp. BYU

CO1233 Lampyridae Lampyrinae Aspisoma sticticum BYU

59 Coll. Code Family Subfamily Species

CO1234 Lampyridae Lampyrinae Cratomorphus sp. BYU

CO1292 Lampyridae Lampyrinae Diaphanes pectinealis BYU

CO1223 Lampyridae Lampyrinae Diaphanes sp. 1 BYU

CO1516 Lampyridae Lampyrinae Diaphanes sp. 2 BYU

CO1524 Lampyridae Lampyrinae Diaphanes sp. 3 BYU

CO1523 Lampyridae Lampyrinae Diaphanes sp. 4 BYU

KSH_6091 Lampyridae Lampyrinae Ellychnia corrusca UGA

CO1197 Lampyridae Lampyrinae Ellychnia sp. BYU

CO1067 Lampyridae Lampyrinae Heterophotinus sp. BYU

CO1294 Lampyridae Lampyrinae Lamprigera yunnana BYU

CO1060 Lampyridae Lampyrinae Lamprocera sp. 1 BYU

CO1459 Lampyridae Lampyrinae Lamprocera sp. 2 BYU

CO1457 Lampyridae Lampyrinae Lamprocera sp. 3 BYU

CO1231 Lampyridae Lampyrinae Lamprocera sp. 4 BYU

CO1287 Lampyridae Lampyrinae Lamprohiza splendidula BYU

CO1071 Lampyridae Lampyrinae Lampyris noctiluca BYU

CO1551 Lampyridae Lampyrinae Lucidota atra BYU

CO1232 Lampyridae Lampyrinae Lucio blattinum BYU

CO1226 Lampyridae Lampyrinae Lychnacris sp. BYU

CO1092 Lampyridae Lampyrinae Microphotus sp. BYU

CO1227 Lampyridae Lampyrinae Petalacmis sp. BYU

CO1549 Lampyridae Lampyrinae Phausis reticulata BYU Phosphaenopterus CO1289 Lampyridae Lampyrinae metzneri BYU

KSH_17651 Lampyridae Lampyrinae Photinus ardens UGA

60 Coll. Code Family Subfamily Species

KSH_1028 Lampyridae Lampyrinae Photinus australis UGA

KSH_9013 Lampyridae Lampyrinae Photinus brimleyi UGA

KSH_9670 Lampyridae Lampyrinae Photinus carolinus UGA

KSH_10061 Lampyridae Lampyrinae Photinus floridanus UGA

KSH_14611 Lampyridae Lampyrinae Photinus granulatus UGA CO1074 Lampyridae Lampyrinae Photinus macdermotti 1 BYU KSH_87151 Lampyridae Lampyrinae Photinus macdermotti 2 UGA CO1547 Lampyridae Lampyrinae Photinus pyralis BYU

CO1224 Lampyridae Lampyrinae Photinus sp. 1 BYU

CO1528 Lampyridae Lampyrinae Photinus sp. 2 BYU

CO1057 Lampyridae Lampyrinae Photinus stellaris BYU CO1230 Lampyridae Lampyrinae Photinini sp. BYU

CO1307 Lampyridae Lampyrinae Pleotomodes needhami BYU

KSH_4081 Lampyridae Lampyrinae Pyractomena borealis UGA

CO1063 Lampyridae Lampyrinae Pyractonema sp. 1 BYU

COARG Lampyridae Lampyrinae Pyractonema sp. 2 BYU

CO1293 Lampyridae Lampyrinae Pyrocoelia pygidialis BYU

CO1552 Lampyridae Lampyrinae Pyropyga decipiens BYU

CO1080 Lampyridae Lampyrinae Pyropyga nigricans BYU

CO1306 Lampyridae Lampyrinae Tenaspis angularis BYU

CO1291 Lampyridae Luciolinae Abscondita cerata BYU Asymmetricata CO1297 Lampyridae Luciolinae circumdata BYU

CO1221 Lampyridae Luciolinae Atyphella flammulans BYU

CO1520 Lampyridae Luciolinae Australoluciola nigra BYU

CO1222 Lampyridae Luciolinae Australoluciola sp. BYU

61 Coll. Code Family Subfamily Species

CO1298 Lampyridae Luciolinae Curtos bilineatus BYU

CO1303 Lampyridae Luciolinae Curtos obsuricolor BYU

CO1255 Lampyridae Luciolinae Curtos sp. BYU

CO1295 Lampyridae Luciolinae Emeia pseudosauteri BYU

CO1220 Lampyridae Luciolinae Lloydiella uberia BYU

CO1518 Lampyridae Luciolinae Luciola sp. 1 BYU

CO1225 Lampyridae Luciolinae Luciola sp. 2 BYU CO1505 Lampyridae Luciolinae Luciolinae nr. Luciola 1 BYU CO1525 Lampyridae Luciolinae Luciolinae nr. Luciola 2 BYU CO1510 Lampyridae Luciolinae Luciolinae nr. Luciola 3 BYU CO1509 Lampyridae Luciolinae Luciolinae nr. Luciola 4 BYU CO1534 Lampyridae Luciolinae Luciolinae sp. BYU

CO1517 Lampyridae Luciolinae Pteroptyx sp. BYU

CO1296 Lampyridae Luciolinae Pygoluciola qinqyu BYU

CO1229 Lampyridae Luciolinae Trisinuata sp. BYU

CO1308 Lampyridae Ototretinae Drilaster sp. BYU

CO1302 Lampyridae Ototretinae Stenocladius shirakii BYU

CO1290 Lampyridae Photurinae Bicellonycha sp. 1 BYU

CO1228 Lampyridae Photurinae Bicellonycha sp. 2 BYU

CO1546 Lampyridae Photurinae Photuris congener BYU

CO1049 Lampyridae Photurinae Photuris divisia BYU

KSH_406 Lampyridae Photurinae Photuris frontalis UGA

KSH_8870 Lampyridae Photurinae Photuris quadrifulgens UGA

CO1062 Lampyridae Photurinae Pyrogaster sp. BYU

CO1550 Lampyridae Pterotinae Pterotus obscuripennis BYU

62 CHAPTER 1 FIGURES

Telegeusis nubifer Microrhagus pygmaeus Cantharidae sp. 1 Cantharidae sp. 2 Calopteron reticulatum Pyrophorus sp. Ampedus pomonae Rhagophthalmus ohbai Phengodes sp. Zarhipis integripennis

86 Lamprigera yunnana A. Luciolinae nr. Luciola 1 Luciolinae nr. Luciola 2 Asymmetrica circumdata Luciolinae sp. Lloydiella uberia Luciola sp. Atyphella flammulans Curtos bilineatus Curtos obsuricolor Australoluciola sp. Australoluciola nigra B. Pteroptyx sp. Trisinuata sp. Abscondita cerata Luciola sp. 2 Luciolinae nr. Luciola 3 95 Emeia pseudosauteri 95 Pygoluciola qingyu Luciolinae nr. Luciola 4 Curtos sp. Pterotus obscuripennis Pollaclasis bifaria C. 72 Stenocladius shirakii 72 Drilaster sp. Phausis reticulata Lamprohiza splendidula Psilocladus sp. 1 72 Psilocladus sigillatus Psilocladus sp.2 Amydetes fastigiata Memoan ciceroi Photuris quadrifulgens Photuris divisia D. Photuris congener Photuris frontalis Bicellonycha sp. 1 Bicellonycha sp. 2 Pyrogaster sp. Vesta impressicollis Vesta saturnalis Vesta sp. Cladodes illigeri Tenaspis angularis Lychnacris sp. E. Petalacmis sp. Lucio blattinum Lamprocera sp. 1 Lamprocera sp. 2 Lamprocera sp. 3 Lamprocera sp. 4 Cratomorphus sp. Pleotomodes needhami Microphotus sp. Diaphanes pectinealis Pyrocoelia pygidialis F. Lampyris noctiluca Diaphanes sp. 1 Diaphanes sp. 2 Diaphanes sp. 3 Luciolinae Diaphanes sp. 4 Pyractomena borealis Pterotinae Aspisoma sp. Aspisoma sticticum Ototretinae Phosphaenopterus sp. Lucidota atra Lamprohizinae Photinini sp. G. Ethra axillaris Heterophotinus sp. Psilocladinae Scissicauda disjuncta Pyropyga decipiens Amydetinae Pyropyga nigricans Pyractonema sp. 1 Photurinae Pyractonema sp. 2 Photinus sp. 1 Lampyrinae Photinus sp. 2 Photinus floridanus Photinus macdermotti 1 H. incertae sedis Photinus stellaris Photinus ardens Photinus carolinus Photinus pyralis 95 Ellychnia sp. Ellychnia corrusca Photinus macdermotti 2 Photinus granulatus Photinus australis Photinus brimleyi 0.3 I. Fig. 1. Maximum Likelihood reconstruction of full, untrimmed dataset aligned in MAFFT; log- likelihood = -26321164.240. Numbers at nodes indicate UFBoot support. Unlabelled nodes have UFBoot values of 100%, colors represent the newly proposed classification. Star at node depicts Lampyridae; A.-I. Dorsal habitus of genus representatives: A. Lamprigera yunnana ; B. Luciola cyathigera; C. Stenocladius davidi; D. Amydetes fastigiata; E. Vesta chevrolati; F. Diaphanes sp.; G. Aspisoma gentile; H. Ellychnia aurora; I. Photinus laticollis;

63

Fig. 2. Lamprohizinae habitus images; A: Phausis reticulata dorsal; B: Phausis reticulata ventral; C: Lamprohiza delarouzei dorsal; D: Lamprohiza delarouzei ventral.

64 *Formatted for submission to Zookeys

Chapter 2: Lampyrid-ID: An interactive LUCID based electronic identification key to the genera

of the Lampyridae (Coleoptera) of the world

Gavin J. Martina,b*, Marc A. Branhamc, Luiz F. L. Da Silveirad,e, Seth M. Bybeea,b a Department of Biology, Brigham Young University, Provo, UT, United States of America b Monte L. Bean Museum, Brigham Young University, Provo, UT, United States of America c Department of Entomology & Nematology, University of Florida, Gainesville, FL, United

States of America d Department of Biology, Western Carolina University, Cullowhee, NC, United States of

America e Department of Plant Biology, University of Georgia, Athens, GA, United States of America

* Corresponding author: [email protected]

Keywords: bioluminescence, fireflies

ABSTRACT

Fireflies (Coleoptera: Lampyridae) are a cosmopolitan group of beetles with a troubled taxonomic history and diverse morphology. Because of this identification resources are lacking.

Here, an electronic Lucid-based identification tool is presented for the genera of Lampyridae

(Coleoptera: Elateroidea). General features used in the key are discussed.

65 INTRODUCTION

Fireflies (Coleoptera: Lampyridae) are a cosmopolitan group of beetles (Branham 2010) that are perhaps most well known for their bioluminescence as adults for most species and larvae for all species (Lloyd 1971, Lewis and Cratsley 2008). This bioluminescence is produced by specialized photic organs normally located on the ventral surface of the abdomen in adults.

While many species of firefly are bioluminescent, there are numerous genera that have no adult light organ. Those that are bioluminescent as adults have a variety of photic organ morphologies

(Branham and Wenzel 2001, 2003). These include light organs that take up entire ventrites and others that appear as isolated stripes across a single ventrite or as various arrangements of spots

(Fig. 1). Fireflies also possess highly diverse antennal morphology. This diversity in morphology occurs not only between species but also between genders. For this reason, several species of firefly are known only from males or females (Barber 1941). While a few identification keys exist for geographical regions (Ballantyne et al. 2019) or for specific tribes (e.g. Silveira et al.

2019), there is no comprehensive resource available for identifying fireflies to genus. This may be a contributing factor to the rampant misclassification of the group (Martin et al. 2019). With current genomic tools, large sets of data are becoming available to elucidate the evolutionary relationships of life on Earth. However, taxonomic expertise in general are becoming more and more rare. This electronic resource will help stem this trend in the fireflies.

66 MATERIALS AND METHODS

Project Description

This identification key is based on the males of the type species (where possible) for each genus and is focused on including as many genera as possible. Several types are either destroyed or reside in unknown locations. For these reasons, we were unable to include several genera at this time.

General Features

The data matrix for this identification key is comprised of 23 morphological and distributional characters with a combined 76 character states. Most characters coded represent external morphological features that do not require dissection or extensive preparation. These characters were derived and/or modified from existing literature (i.e. Olivier 1907, McDermott

1966, Jeng 2008). When possible, scorings were confirmed by examination of type and other material, comprised of several specimens for each genus with an emphasis on the type species.

Specimens were examined from several museums: California Academy of Sciences (CAS),

Sacramento, CA, USA; Florida State Collection of Arthropods (FSCA), Gainesville, FL, USA;

National Museum of Natural History (USNM), Washington D.C., USA; Australian National

Insect Collection (ANIC), Canberra, Australia; Muséum National d'Histoire Naturelle (MNHN),

Paris, France; The Natural History Museum (NHM), London, United Kingdom;

Naturhistorisches Museum (NHMB), Basel, Switzerland; and Coleção Entomológica Prof. José

Alfredo Pinheiro Dutra (DZRJ), Rio de Janeiro, Brazil.

List of characters used in key

Geographic distribution;

Number of ventrites (six/seven/eight);

67 Antennal features:

Antenna (filiform/serrate/uniflabellate/biflabellate/highly modified, terminal

antennomere elongate, longer than all preceding antennomeres);

Sensorum on apical antennomere (absent/present);

Relative length of scape/antennomere 1 (longer than pedicel (antennomere 2)/shorter than

pedicel (antennomere 2));

Number of antennomeres (fewer than 11/11/12/17 to 18/more than 22);

Head features:

Eye separation in ventral aspect (widely separated (interocular distance wider than width

of eye)/moderately separated but not touching(interocular distance narrower than width

of eye)/narrowly separated to contiguous);

Mandibles (large and arcuate/moderately sized and arcuate/small and needle-

shaped/highly reduced/not visible);

Number of labial palpomeres (4/3/2/1);

Thoracic features:

Anterior margin of pronotum (flat, not reflex/weakly reflexed/strongly reflexed);

Prothoracic trochantin (glabrous/setose);

Leg features:

Hindleg tibial spurs (absent/2 small spurs (barely longer than surrounding setae)/2 large

spurs);

Tarsal pad (absent/present on tarsomere 4);

Hindleg claws (apically entire/both claws bifid apically/one claw bifid apically);

Metafemoral comb (absent/present);

68 Elytral features:

Elytral costae (absent/present);

Apex of elytra (more or less independently rounded/somewhat acute/with ventral back-

folds);

Abdominal features:

Ventrite 6, if last ventrite, median posterior projection (absent/prolonged into paired,

asymmetric hooks/present);

Ventrite 7, posterior pointed projection (absent/present);

Abdominal spiracles (ventral/dorsal).

Light organ features:

Light organ on abdominal ventrite 5 (absent/fully occupying ventrite 5/a

transverse stripe/a large, transversely elliptical central spot/a small round spot);

Light organ on abdominal ventrite 6 (absent/occupying full or nearly full

ventrite/bipartate/restricted to anterior portion of ventrite/confined to center of

ventrite/two small round spots/);

Light organ on abdominal ventrite 7 (absent/present, two small lateral spots);

Software technical specifications

Application: LucidTM Builder 3.3 (www.lucidcentral.org)

Key version: 1.0

Requirements for use: Java-enabled browser and internet connectivity

License for use of the key: Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided original author and source are credited

69 Web location: https://keys.lucidcentral.org/keys/v3/lampyridae/

70 CONCLUSIONS

Fireflies have a long and convoluted taxonomic history. Many genera were described inadequately in the late 1800’s and early 1900’s (Olivier 1907). This has made understanding generic limits and identification of Lampyridae difficult. This identification key, based primarily on the type species for each genus, should facilitate the identification of specimens to genus and advance future studies of the family Lampyridae. Future taxonomic efforts will surely describe currently unknown biodiversity as well as further refine our concepts of taxonomic groups within the family Lampyridae. It is our hope that this identification key to genera will be expanded and refined to integrate such contributions. Several known genera have yet to be included in this identification key or are coded from species other than the type species. This is either due to the type species/specimens being lost (e.g. Anadrilus), or extremely rare (e.g. Cyphonocerus). Future editions of this identification key will include as many of these genera as possible, while also working to improve the morphological character coding used to support identification.

71 ACKNOWLEDGEMENTS

We greatly appreciate the curators and collection managers who provided specimens critical to this study: Cate Lemann (ANIC), Chris Grinter (CAS), Paul Skelley and Kyle Schnepp

(FSCA), Azadeh Taghavian (MNHN), Max Barclay and Michael Geiser (NHM), Isabelle

Zuercher (NHMB), and Floyd Shockley (USNM). We also thank Gareth Powell, Robert

Erickson, and Kristin Dunn for assistance on collection visits. We thank Oliver Keller, Natalie

Saxton and the Bybee Lab group for preliminary testing of identification resources. This work was funded by NSF: DEB-1655981 (SMB), DEB-1655936 (MAB), and DEB-1655908 (awarded to Kathrin Stanger-Hall in support of L.F.L.D.S.).

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74 CHAPTER 2 TABLES

Table 1: Genera and species used for coding of key-matrix. Type species in bold. Genus/Terminal taxa Source of coding Museum Subfamily Abscondita Ballantyne, Lambkin, and Abscondita cerata USNM Luciolinae Fu in Ballantyne et al., 2013 Afrodiaphanes Afrodiaphanes Geisthardt, 2007 NHM Lampyrinae marginipennis Alecton Laporte, 1833 Alecton discoidalis USNM Lampyrinae Amydetes Hoffmannsegg in Illiger, Modified from Jeng, N/A Amydetinae 1807 2008 Ankonophallus Zaragoza-Cabellero Ankonophallus N/A Lampyrinae and Navarrete-Heredia, 2014 zuninoi Aorphallus Zaragoza-Caballero and Aorphallus cibriani N/A Lampyrinae Gutierrez-Carranza, 2018 Aquatica Fu, Ballantyne, and Aquatica ficta USNM Luciolinae Lambkin, 2010 Aquatica hydrophila USNM Aquilonia Ballantyne in Ballantyne Aquilonia costata ANIC Luciolinae and Lambkin, 2009 Araucariocladus Silveira and Araucariocladus hiems Personal Incertae sedis Mermudes, 2017 Aspisoma Laporte, 1833 Aspisoma dilatatum USNM Lampyrinae Aspisoma ignitum USNM Aspisoma perplexum USNM Aspisoma sticticum USNM Aspisomoides Zaragoza-Caballero, Aspisoma bilineatum USNM Lampyrinae 1995 Asymmetricata Ballantyne in Asymmetricata USNM Luciolinae Ballantyne and Lambkin, 2009 circumdata Asymmetricata ovalis USNM Atyphella Olliff, 1890 Atyphella atra ANIC Luciolinae Atyphella guerini ANIC Atyphella inconspicua ANIC Atyphella lewisi ANIC Atyphella similis ANIC Australoluciola Ballantyne in Australoluciola ANIC Luciolinae Ballantyne and Lambkin, 2013 flavicollis Bicellonycha Motschulsky, 1853 Bicellonycha amoena USNM Photurinae Bicellonycha USNM cyathigera Bicellonycha USNM ornaticollis Modified from Jeng, Brachylampis Van Dyke, 1939 N/A Ototretinae 2008

75 Callopisma Callopisma Motschulsky, 1853 USNM Lampyrinae dimidiatipennis Callopisma ramsdeni USNM Callopisma rufa USNM

Calyptocephalus Gray in Griffith and Calyptocephalus USNM Lampyrinae Pidgeon, 1832 opimus Modified from Jeng, Ceylanidrilus Pic, 1911 N/A Ototretinae 2008 Modified from Jeng, Cheguevaria Kazantsev, 2006 N/A Cheguevariinae 2008 Cladodes Solier in Gay, 1849 Cladodes demoulini USNM Lampyrinae Cladodes flabellatus USNM Cladodes lamellicornis USNM Colophotia Motschulsky, 1853 Colophotia concolor USNM Luciolinae Colophotia praeusta USNM Convexa Ballantyne in Ballantyne and Convexa wolfi ANIC Luciolinae Lambkin, 2009 Crassitarsus Martin in Martin et al. Pachytarsus bryanti USNM Incertae sedis 2019 Cratomorphus Cratomorphus Motschulsky, 1853 USNM Lampyrinae cossyphinus Cratomorphus USNM diaphanus Curtos Motschulsky, 1845 Curtos okinawanus USNM Luciolinae Modified from Jeng, Cyphonocerus Kiesenwetter, 1879 N/A Cyphnocerinae 2008 Dadophora Olivier in Wytsman, 1907 Dadophora hyalina NHM Lampyrinae Diaphanes Motschulsky, 1853 Diaphanes pellucens USNM Lampyrinae Dilychnia Motschulsky, 1853 Dilychnia guttala USNM Lampyrinae Dodacles Olivier, 1885 Dodacles emissa USNM Lampyrinae Dodacles plumosa USNM Drilaster Kiesenwetter, 1879 Ototreta bakeri USNM Ototretinae Ototreta bicoloripes USNM Ototreta fornicatus USNM Dryptelytra Dryptelytra Laporte, 1833 USNM Lampyrinae cayennensis Dryptelytra fulvipennis USNM Ellychnia Blanchard, 1845 Ellychnia californica USNM Lampyrinae Modified from Jeng, Erythrolychnia Motschulsky, 1853 N/A Lampyrinae 2008 Modified from Jeng, Ethra Laporte, 1833 N/A Lampyrinae 2008

76 Modified from Jeng, Falsophaeopterus Pic, 1911 N/A Ototretinae 2008 Flabellototreta Pic, 1911 Flabellotreta inapicalis USNM Ototretinae Gorhamia Gorhamia Pic, 1911 USNM Ototretinae compressicornis Harmatelia Walker, 1858 Harmatelia bilinea USNM Ototretinae Inflata Boontop in Ballantyne et al., Inflata indica ANIC Luciolinae 2015 Modified from Jeng, Lamellipalpodes Maulik, 1921 N/A Ototretinae 2008 Lamprigera Motschulsky, 1853 Lamprigera boyei USNM Incertae sedis Lamprocera Laporte, 1833 Lamprocera diluta USNM Lampyrinae Lamprocera extincta USNM Lamprocera latreillei USNM Lamprocera torquata USNM Lamprohiza Motschulsky, 1853 Lamprohiza delarouzei USNM Lamprohizinae Lamprohiza USNM splendidula Lampyris Geoffroy, 1762 Lampyris noctiluca USNM Lampyrinae Modified from Jeng, Lampyroidea Costa 1875 N/A Luciolinae 2008 Ledocas Olivier, 1885 Ledocas scutellatus USNM Lampyrinae Lloydiella Ballantyne in Ballantyne Lloydiella majuscula ANIC Luciolinae and Lambkin, 2009 Modified from Jeng, Lucernuta Laporte, 1833 N/A Lampyrinae 2008 Lucidina Gorham, 1883 Lucidina accensa USNM Lampyrinae Lucidina biplagiata USNM Lucidota Laporte, 1833 Lucidota atra USNM Lampyrinae Lucidota bella USNM Modified from Jeng, Lucidotopsis McDermott, 1960 N/A Lampyrinae 2008 Modified from Jeng, Lucio Laporte, 1833 N/A Lampyrinae 2008 Modified from Jeng, Luciola Laporte, 1833 N/A Luciolinae 2008 Luciuranus Silveira, Khattar, and Luciuranus josephi Personal Lampyrinae Mermudes in Silveira et al., 2016 Lychnacris Motschulsky, 1853 Callopisma emarginata Lampyrinae Modified from Jeng, Macrolampis Motschulsky, 1853 N/A Lampyrinae 2008 Magnalata Ballantyne in Ballantyne Modified from Jeng, N/A Luciolinae and Lambkin, 2009 2008 Magnoculus McDermott, 1964 Magnoculus costatus USNM Amydetinae

77 Magnoculus USNM guatemalae Medeopteryx Ballantyne in Ballantyne Medeopteryx effulgens ANIC Luciolinae and Lambkin, 2013 Medeopteryx cribellata USNM Memoan Silveira and Mermudes, 2013 Memoan ciceroi USNM Amydetinae Microdiphot Barber, 1941 Microdiphot barberi USNM Lampyrinae Micronaspis Green, 1948 Micronaspis floridana USNM Lampyrinae Microphotus LeConte, 1866 Microphotus dilatatus USNM Lampyrinae Modified from Jeng, Mimophotinus Pic, 1935 N/A Lampyrinae 2008 Nelsonphotus Cicero, 2006 Nelsonphotus aridus FSCA Lampyrinae Nyctophila Olivier, 1884 Nyctophila incisa NHM Lampyrinae Oculogryphus Jeng, Engel, and Yang, Oculogryphus shuensis USNM Incertae sedis 2007 Modified from Jeng, Ophoelis Olivier, 1911 N/A Lampyrinae 2008 Modified from Jeng, Ototretadrilus Pic, 1921 N/A Ototretinae 2008 Pacifica Ballantyne in Ballantyne and Pacifica limbatipennis USNM Luciolinae Lambkin, 2013 Pacifica salomonis USNM Modified from Jeng, Paraphausis Green, 1949 N/A Lampyrinae 2008 Pelania Mulsant, 1860 Pelania mauritanica USNM Lampyrinae Petalacmis Olivier, 1908 Petalacmis praeclarus MNB Lampyrinae Phaenolis Gorham, 1880 Phaenolis laciniatus USNM Lampyrinae Phaenolis ochracea USNM Phaenolis ustulatus USNM Phausis LeConte, 1852 Phausis inaccensa USNM Lamprohizinae Phausis reticulata USNM Phausis riversi USNM Phosphaenopterus Phosphaenopterus Schaufuss, 1870 USNM Lampyrinae montandoni Phosphaenus Phosphaenus Laporte, 1833 USNM Lampyrinae hemipterus Photinoides McDermott, 1963 Photinoides penai USNM Lampyrinae Photinus Laporte, 1833 Photinus blackwelderi USNM Lampyrinae Photinus carolinus USNM Photinus congruus USNM Photinus USNM consanguineus Photinus consimilis USNM Photinus euphotus USNM Photinus pallens USNM

78 Photinus pyralis USNM Photoctus McDermott, 1961 Photoctus boliviae USNM Incertae sedis Photuris Dejean, 1833 Photuris hebes USNM Photurinae Photuris pennsylvanica USNM Photuris versicolor USNM Modified from Jeng, Picodrilus Wittmer, 1938 N/A Ototretinae 2008 Modified from Jeng, Platylampis Motschulsky, 1853 N/A Lampyrinae 2008 Modified from Jeng, Pleotomodes Green, 1948 N/A Lampyrinae 2008 Pleotomus LeConte, 1861 Pleotomus davisii USNM Lampyrinae Pleotomus nigripennis USNM Pleotomus pallens USNM Pollaclasis Newman, 1838 Pollaclasis bifaria USNM Incertae sedis Presbyolampis Presbyolampis Buck, 1947 USNM Photurinae immigrans Modified from Jeng, Pristolycus Gorham, 1883 N/A Luciolinae 2008 Modified from Jeng, Pseudolychnuris Motschulsky, 1853 N/A Lampyrinae 2008 Psilocladus Blanchard, 1846 Psilocladus guerini USNM Psilocladinae Pteroptyx Olivier, 1902 Pteroptyx tener USNM Luciolinae Pygatyphella Ballantyne, 1968 Atyphella obsoleta ANIC Luciolinae Pterotus LeConte, 1859 Pterotus obscuripennis USNM Pterotinae Pygoluciola Wittmer, 1939 Pygoluciola qingyu ANIC Luciolinae Pyractomena Melsheimer, 1846 Pyractomena lucifera USNM Lampyrinae Pyractonema Solier in Gay, 1849 Pyractonema angustata USNM Lampyrinae Pyractonema latior USNM Pyrocoelia Gorham, 1880 Pyrocoelia lateralis USNM Lampyrinae Pyrocoelia plagiata USNM Pyrocoelia praetexta USNM Pyrocoelia rufa USNM Pyrogaster Motschulsky, 1853 Pyrogaster lunifer USNM Photurinae Pyrogaster squalidus USNM Pyrogaster USNM telephorinus Pyrophanes Olivier, 1885 Pyrophanes beccarii USNM Luciolinae Pyropyga Motschulsky, 1852 Pyropyga decipiens USNM Lampyrinae Pyropyga fenestralis USNM (nigricans) Pyropyga minuta USNM Modified from Jeng, Robopus Motschulsky, 1853 N/A Lampyrinae 2008 Roleta McDermott, 1962 Roleta coracina USNM Lampyrinae

79 Modified from Jeng, Rufolychnia Kazantsev, 2006 N/A Lampyrinae 2008 Scissicauda McDermott, 1964 Scissicauda disjuncta MNHN Lampyrinae Sclerotia Ballantyne in Ballantyne et Sclerotia aquatilis ANIC Luciolinae al., 2016 Stenocladius Fairmaire in Deyrolle Stenocladius basalis USNM Ototretinae and Fairmaire, 1878 Tenaspis LeConte, 1881 Tenaspis angularis USNM Lampyrinae Tenaspis sinuosa USNM Triangulara Pimpasalee in Ballantyne Triangulara ANIC Luciolinae et al., 2016 frontoflava Uanauna Campello-Goncalves, Souto, Uanauna angaporan Personal Lampyrinae Mermudes, and Silveira, 2019 Vesta Laporte, 1833 Vesta basalis USNM Lampyrinae Vesta despecta USNM Ybytyramoan Silveira and Mermudes, Ybytyramoan NHM Lampyrinae 2014 monteirorum

80 CHAPTER 2 FIGURES

A. B. C. D. E.

F. G. H. I. J. Figure 1: Ventral habiti of various species showing diversity of photic organ and antennal morphology. A. Magnalata rennelia; B. Pyractomena vexillaria; C. Luciola amplipennis; D. Photuris excavaticeps; E. Dilychnia disparilis; F. Cladodes illigeri; G. Lucidota aurantiaca; H. Vesta rustica; I. Psilocladus pulcher; J. Amydetes fastigiata

81 *Formatted for submission to BMC Evolutionary Biology Chapter 3: The genes of the phototransduction pathway among fireflies (Coleoptera:

Lampyridae)

Gavin J. Martin1,2,*, Sarah E. Lower3, Anton Suvorov1,4, Seth M. Bybee1,2

1Department of Biology, Brigham Young University, Provo, UT, United States

2Monte L. Bean Museum, Brigham Young University, Provo, UT, United States

3Department of Biology, Bucknell University, Lewisburg, PA, United States

4Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC United

States

*Corresponding Author: [email protected]

Keywords: Arrestin-2, inaC, positive selection, vision, sensory niche

ABSTRACT

Background:

Most organisms are dependent on sensory cues from their environment for survival and reproduction. Fireflies (Coleoptera: Lampyridae) represent an ideal system for studying sensory niche adaptation due to many species relying on bioluminescent communication, as well as a diversity of ecology. Here, using transcriptomics, we examine the phototransduction pathway in this non-model organism, and provide some of the first evidence for positive selection in the PT pathway beyond opsins in beetles.

Results:

82 Gene duplications are found in calmodulin, inactivation no afterpotential C, inactivation no afterpotential D, and transient receptor potential. We also find strong support for positive selection in arrestin-2, inactivation no afterpotential D, and transient receptor potential, with weak support for positive selection in guanine nucleotide-binding protein G(q) subunit alpha and neither inactivation nor afterpotential C.

Conclusions:

Our results show positive selection in certain genes in diurnal fireflies. This is one of the first times positive selection is investigated in the beetle phototransduction pathway. Taken with other recent work in flies, butterflies, and moths, this represents an exciting new avenue of study as we seek to further understand diversification and constraint on the PT pathway in light of organism ecology.

83 INTRODUCTION

Background

Organisms rely on sensory input from the environment to inform their basic ecology.

Inputs occur across different channels, such as auditory (e.g. song), tactile (e.g. mechanosensory stimulation), gustatory (e.g. tastes), olfactory (e.g. pheromones, kairomones), and visual (e.g. bright colors, behavioral displays) channels, and are detected by different sensory structures and underlying molecular pathways. While many animals integrate input from several sensory channels depending on the circumstances, often a single channel dominates (e.g. vision in primates: [1], dragonflies and damselflies [2]), perhaps due to tradeoffs in investment in sensory structures [3, 4] and the interplay of selection and constraint on the underlying molecular players

(e.g. [5]). The extent to which selection and constraint play a role in adaptation to an organism’s specific sensory niche, particularly with respect to the underlying molecular mechanisms, remains a fascinating question that has been investigated mostly by work in model systems.

Modern sequencing technologies enable interrogation of these questions in non-model organisms, which present unique opportunities to study evolutionary forces in systems where evolutionary changes in primary sensory niche (i.e. nocturnal vs. diurnal) are known.

Fireflies (Coleoptera: Lampyridae) are an excellent system for studying sensory niche adaptation due to variation in their conspicuous bioluminescent mating displays. With over 2000 species distributed around the globe, variation in flash patterns across nocturnal taxa is diverse and serves in both species recognition and mate choice [6, 7]. While the proximate and ultimate reasons for signal diversity in light-using species have been studied for centuries [8–11], many species have lost the ability to produce light as adults. Instead of light, these day-active fireflies

84 rely on pheromone signals to locate, recognize, and choose mates [12–14]. In contrast, pheromones may be relatively unimportant for nocturnal light-using species [12]. The change from nocturnal, primarily-visual, light signal use to diurnal, primarily-pheromone, signal use, makes fireflies an ideal system for testing hypotheses about the role of selection in these evolutionary transitions in sensory niche.

Previous studies in fireflies highlight the interplay of diversification and constraint in both morphological and molecular adaptations to sensory niche. Nocturnal, light-using fireflies that rely on bioluminescence for communication have larger eyes and smaller antennae than their diurnal, pheromone-using relatives [4]. This inverse relationship between eye and antenna size across sensory niches may indicate developmental constraints that limit investment in antenna at the expense of eyes, and vice versa [15]. At the molecular level, previous research has demonstrated that contrary to the insect eye bauplan, fireflies along with most other beetles and their close relatives have only two opsin copies: one ultraviolet and one long-wavelength [16–

18]. [17] found evidence for positive selection in the long-wavelength opsins among diurnal species, but not nocturnal. Additionally, certain fireflies have been shown to vary the expression levels of the long-wavelength opsin in correlation with peak bioluminescent signaling time [19].

However, opsins represent only one of many molecular players in the phototransduction pathway, begging the question - are changes in other members of the pathway associated with evolutionary transitions in sensory niche?

Vision in fireflies, like all insects, is regulated by the components of the phototransduction (PT) pathway (Figure 1). The phototransduction pathway consists of several

85 key genes and is essentially a seven to eight step process that starts with light activating the insect rhodopsin and is terminated by arrestin-1 and/or -2 binding to the rhodopsin (for a more complete review see [20]. While most of what is known about the insect PT pathway comes from work in the Dipteran fruit fly model organism, Drosophila melanogaster, and more recently the

Lepidopeteran Heliconius [20] little is known about the pathway in other insect groups, with the exception of opsins [21–23]. Opsins have also been well studied in the fireflies [16, 17]. Further, exploring the full phototransduction pathway in fireflies provides one of the first studies of this pathway with respect to signal niche adaptation and potential for selection in insects.

With growing genomic tools for non-model organisms, including fireflies [17, 24], it is possible to examine the entire PT pathway and form hypotheses regarding the visual evolution of these organisms. Here, we search publicly available and newly-sequenced firefly transcriptomes

(20 taxa in total) to identify the visual pathway genes and address the following objectives: 1)

Identify genes involved in phototransduction in firefly transcriptomes; 2) Examine patterns of duplication and loss as compared to other model insects (i.e. Drosophila and Tribolium); and 3)

Test hypotheses of selection related to sensory niche adaptation (nocturnal vs diurnal). The reliance on bioluminescent communication to find mates for many, but not all, adult fireflies provides an ideal and simple system to study the evolution of both the visual signal (adult bioluminescence) and receiver system (adult eyes).

86 MATERIALS AND METHODS

Sampling and Sequencing

Transcriptomes utilized in this study were taken from [16] and [17]. RNA was extracted from several body parts (i.e. head only, abdomen/light organ only, and whole body). As such, while there are 32 RNA-seq libraries in the study, these represent 20 taxa (Table 1, datadryad.org/stash/share/QRYn_7voIXFgtWFQH6E398eIkEfRW-ZicXx5dHjbWJg).

Both head and body tissue for transcriptome assembly was prepped separately for the following taxa: Micronaspis floridana Green, Pyractomena dispersa Green, P. pyralis Linnaeus

(see [16]). Total RNA was extracted from each taxon using NucleoSpin columns (Clontech) and reverse-transcribed into cDNA libraries using the Illumina TruSeq RNA v2 sample preparation that both generates and amplifies full-length cDNAs. Prepped mRNA libraries were sequenced on an Illumina HiSeq 2000 utilizing 101-cycle paired-end reads by the Microarray and Genomic

Analysis Core Facility at the Huntsman Cancer Institute at the University of Utah, Salt Lake

City, UT, USA.The transcriptomic data allows us to combine current data, as well as gain a knowledge of what parts of the phototransduction pathway are actually expressed in the firefly eye.

Transcriptome assembly and annotation

Quality control, assembly, and transcriptome searches were performed using a pipeline constructed from existing computational tools after [60]to facilitate downstream evolutionary analyses. In short, RNA-seq reads were trimmed using the Mott algorithm implemented in

PoPoolation version 1.2.2 [61], with a minimum read length=40 and quality threshold=20. The

87 de novo assembly of the transcriptome contigs was carried out using Trinity version 2.0.6 [62] under the default parameters. Assembled transcriptomes were then annotated using the trinotate release 2013-08-26 pipeline. Detection and filtering for putative phototransduction gene sequences from the Lampyridae transcriptomes were performed against a database using insect visual phototransduction homologous groups from ORTHODB database [63]. Recovered insect sequences from the PT pathway were then aligned using MAFFT v. 7.407 [64] and converted into a profile hidden Markov model (pHMM) database using hmmbuild program of HMMER

3.1b1 [65]. Using this database we screened TransDecoder-predicted proteomes for genes known from the PT pathway against pHMM database using hmmscan with an e-value cutoff of 10-5.

Additionally, we used PIA [66] with default parameters to identify phototransduction genes that may be missed by the HMMER search using the raw Trinity transcriptome assemblies. All redundant (identical) phototransduction gene sequences identified by both approaches were removed using CDHIT [67, 68]. After all putative hits were compiled, sequences were screened for any false positives, including sequences coding for other genes, or duplicate reads not the result of biology, but rather read coverage. Sequences were aligned via MAFFT to remove sequences with >98% similarity. Each unique sequence was then Blast searched using megablast against the NCBI database to confirm gene identity.

Analyses of positive selection

Sequences for each gene were aligned independently in MAFFT using the default automatic alignment option. Each subsequent alignment was used for Maximum Likelihood tree inference implemented within IQ-Tree v. 1.6.8 [69]. Tests for positive selection in each gene were performed in PAML v. 4.9 [26]. Using the branch-specific test, the free-ratios model (Ha;

88 model=2, nsites=0) was tested against the branch model (Ho; model=0, nsites=0). The log likelihood of each model was compared with the likelihood ratio test (LRT) using �2 distributions (3.84 at p-value=0.05) with appropriate degrees of freedom.

Inference of positive selection has been shown to be greatly influenced by the accuracy of

MSA [28]. In particular the inference of selection obtained on MSAs produced by MAFFT shows overconfidence in identification of positive selection (i.e. increased rates of false positive results). In order to mitigate that problem we implemented a Bayesian approach utilized in BAli-

Phy version 3.4.1 [27] that essentially jointly estimates MSAs and positive selection (in our case branch-specific) and exhibits superior accuracy [28]. To run analyses of branch positive selection in BAli-Phy, for each gene we used the corresponding tree estimated by ML initializing 3 independent MCMC chains with 3000 iterations each as in [28]. MSA was sampled every 5th iteration. The seemingly low number of iterations is explained by the fact that at each MCMC draw BAli-Phy updates multiple parameters compared to other MCMC software where only one parameter is updated per iteration. In order to improve our estimator of positive selection we used Rao-Blackwellization technique, i.e. taking a conditional expectation of the current estimator.

The results of 3 runs were pulled together discarding 15% burn-in for each gene. Then

Bayes factors (BF) were calculated to assess support for positive selection. We followed a scoring scheme proposed by [70], where BF > 20 exhibits strong support for positive selection,

20 < BF < 3 exhibits “positive” support and BF < 3 is “not worth more than a bare mention”.

89 RESULTS

Transcriptome assemblies recover most conserved genes:

To examine patterns of gene duplication and test for selection in the PT pathway across firefly species, we compiled transcriptome assemblies from published and new datasets.

Transcriptomes varied in quality, generally according to input tissue type (Table 1).

Transcriptome assemblies captured the majority of genes as full-length transcripts - mean

BUSCO completeness across assemblies was 76% (range: 40–95%), the mean N50 was 2,096 bp, and the mean maximum contig length was 17,138 bp. Total number of contigs varied from

19,676 to 77, 811. In general, transcriptomes derived from the whole body or the head region were longer and more complete than those derived from abdominal tissue only (Table 1).

Orthologous search strategy identifies duplications in phototransduction genes:

Several gene families in the PT pathway have been shown to contain duplication events within insects [25]. Genes identified in this dataset by orthologous search, followed by manual curation of results, included arrestin-1 (arr1), arrestin-2 (arr2), calmodulin (Cam), guanine nucleotide-binding protein G(q) subunit alpha (Gq), G protein-coupled receptor kinase 1

(Gprk1), inactivation no afterpotential C (inaC), inactivation no afterpotential D (inaD), inactivation no afterpotential E (inaE), neither inactivation nor afterpotential C (ninaC), no receptor potential A (norpA), transient receptor potential (trp), and transient receptor potential- like (trpl). Unfortunately, arr1 and trp were only recovered from three taxa, none of which were diurnal. Therefore we were unable to test the genes further. While not every gene was found in every species (likely due to the incompleteness of the data), putative duplications of various genes were found in several species: Photinus pyralis (Cam & inaC), Photinus australis (Cam),

90 Photinus macdermotti (inaC), Lucidota atra (inaC), Phausis reticulata (inaC), Bicellonycha wickershamorum (inaC), and Photinus carolinus (inaD); see Table 2. These putative duplicates were confirmed in the Photinus pyralis genome [24], however the putative duplicate of inaD within Photinus carolinus does not seem to be present in the P. pyralis genome, and therefore requires further confirmation.

Analysis for positive selection identifies genes under positive selection:

PAML: Tests for positive selection were performed in PAML [26] on two sets of trees resulting from two different approaches to alignment. First, PAML was run on the original

MAFFT alignment. The branch model test (model=0, nsites=0) for the null hypothesis showed elevated omega values (average ⍵ across all genes 0.076) for Gq (⍵=0.114), inaD (⍵=0.114), ninaC (⍵=0.110), and trpl (⍵=0.230). This analysis also resulted evidence for positive selection

(lrt > 3.84 at p value = 0.05) in arr2 (lrt=43.59), inaC (lrt=64.35), inaD (lrt=28.04), ninaC

(lrt=43.76), and trpl (lrt=16.07) when the alternative hypothesis (model=2, nsites=0) was selection in diurnal lineages. Second, PAML was run on the alignment resulting from BAli-Phy

[25, 27] to account for potential overestimation of positive selection in the MAFFT alignment

[28]. This analysis resulted in elevated omega values (average ⍵ across all genes 0.046), inaD

(⍵=0.092), ninaC (⍵=0.109), and trpl (⍵=0.082). The PAML analysis of the BAli-Phy alignment also resulted in support for evidence of positive selection in arr2 (lrt=56.28), Gq

(lrt=5.02), inaD (lrt=22.30), ninaC (lrt=45.10), and trpl (lrt=114.44); (Table 3).

Consistent between the two PAML results arr2, inaD, ninaC, and trpl were identified as possibly evolving under positive selection.

91 BAli-Phy: To further explore genes identified by PAML to be under positive selection, we also tested for genes under positive selection using BAli-Phy by examining the Bayes Factor (BF).

The genes arr2 (BF: 9.5), Gq (82.28), inaD (5.13), and trpl (8.59) were again identified as evolving under positive selection (Figure 2; Table 3)

Given these results, there is strong evidence in all three analyses for arr2, inaD, and trpl being under positive selection in diurnal lineages, with additional support in two analyses for positive selection in ninaC and Gq (Table 3).

92 DISCUSSION

The insect phototransduction cascade is most well-known and studied in the model insects Drosophila and Heliconius (reviewed in [29][20]). Here we increase the knowledge of this cascade system by exploring the PT pathway in fireflies. We further explore these data by looking for patterns of selection that are potentially related to the signaling ecology of nocturnal vs diurnal fireflies.

Duplication rates of phototransduction genes have been shown to be higher in arthropods and related groups as opposed to other metazoan organisms [25]. These duplications have been shown in several gene families such as TRP, r-opsin, and arrestin among others [20, 25]. Here we add to the known duplication events in insects by showing evidence for duplications in Cam, inaC, and inaD. What is especially interesting about these findings is the lack of inaC within the

Tribolium genome [30]). While functionally similar paralogues where found in Tribolium (one of which is likely homologous to the inaC copy recovered in this study), there is quite a divergent biology between the cereal pest Tribolium and the fireflies. InaC was recovered in the phototransduction survey of the blind, cave-dwelling Ptomaphagus hirtus (Coleoptera:Leiodidae

[31]). This suggests that the inaC gene was likely lost after the divergence of the Staphilinoidea with remaining polyphagan beetles [32]. Future research is needed to determine exactly when this loss occurred, and with which sensory niche the loss is associated. The duplication in the

Cam gene was recovered in several firefly lineages and seems to be closely allied with a yet uncharacterized neo-calmodulin like gene.

93 In insect phototransduction, inaD acts a scaffold protein that is central to a group of proteins commonly known as the signalplex [33–37]. This signalplex is composed primarily of inaD, trp, norpA, and inaC, also found in one of our analyses to be under positive selection [29].

If inaD is lost or disrupted, it causes a downstream breakup of the signalplex [38]. Further, inaD stability is dependent upon trp, a Ca2+ -permeable cation within the cell [29, 39, 40]. Further upstream in the cascade Gq plays a role in the portion of the cascade that leads to the production of DAG and MAG [41] which are hypothesized to play a critical role in activation of trp and trpl

[20, 42].

The interaction between trp and trpl in organisms which inhabit low-light vs. high-light environments has received much recent attention. It appears that one or the other can be up- or down-regulated depending on the dominant light environment. In Drosophila melanogaster

(active primarily during the day) trp is not only the more abundant channel, but also flies act blindly with mutated trp [43]. In cockroaches (primarily nocturnal) the opposite was shown, trpl was the more abundant channel by far [44]. Recently, Macias-Munoz and colleagues. [20] have shown an interesting relationship within the Lepidoptera. The day active Heliconius melpomene showed no differentiation in trp vs trpl channels, however the night-active Manduca sexta showed down-regulation of trp, showing a similar pattern to that of the cockroaches [20]. Here, we show strong support for the hypothesis that trpl is under more positive selection in the diurnal fireflies Ellychnia and Lucidota.

InaD also forms a protein complex with ninaC (also recovered in two of the three tests for positive selection), each of them binding Cam. This complex has been shown to mediate Ca2+

94 movement, thus greatly increasing the efficiency of arrestin [45, 46]. In order for arrestin movement to take place arrestins must bind to phosphoinositides, which is thought to be mediated via ninaC ([47], however see [48]).

There is also strong support for the detection of positive selection in arr2 in diurnal species of fireflies. This is not surprising given the critical role of arr2 in light-mediated signaling. The arrestin gene family is well known for mediation in many G protein-coupled receptor signaling cascades [49]. In these systems, its purpose is to arrest or stop signaling [50,

51]. While there are four known arrestin genes in mammalian systems [52], only two, arrestin-1 and arrestin-2 have been found in Drosophila [53–56]. Both arrestin genes were expressed in our

Lampyridae taxa, however arrestin-1 was only found in a handful of taxa, and not recovered in either of the diurnal species included. In Lepidoptera, it has been shown that arr2 is more highly expressed than arr1. Arr2 does not reside in the rhabdomere (where phototransduction takes place) but is trafficked into the rhabdomere after phototransduction (see above).

95 CONCLUSIONS

This study represents one of the first attempts to assess positive selection in the phototransduction genes in a non-model insect beyond the r-opsin gene family. Arr2, inaD, and trpl represent critical components of the insect phototransduction pathway. All three are associated with light-dependent interactions within the eye. While sexual communication in many fireflies is critically dependent on vision, bioluminescent communication tends to take place beginning in the dusk hours and continuing into the night. In this dark environment, regardless of sexual activity, there are simply fewer photons reaching the firefly eye, meaning the PT pathway is not as active. For those lineages (Ellychnia and Lucidota) active during the day, and therefore more dependent on light for survival and reproduction, it is intriguing that these light-activated genes have support for evolving under positive selection, and are therefore diverging from those in the conserved, nocturnal sensory niche.

As we investigate the variation in this system, both with a larger taxon sampling, and with more complete genomic resources, our understanding of insect vision will come into sharper focus. While this study adds an important component in insect visual research of non- model organisms, much more remains to be addressed. For example, an in depth quantification and qualification of PT genes across multiple firefly genomes, as well as expression data from throughout the diel changes to compare potential differences of PT genes expression. Such experiments would also provide insight into dimorphic gene expression patterns as has been found in one species of firefly [19]. Additionally, there is more variation in firefly communication systems that could be investigated. These modes of communication have undergone various classification systems [57, 58] however most recently firefly communication

96 modes were simplified into four groups: pheromone use only, continuous glow + pheromone, short or long flashes, and pheromone use + weak, daylight glow [59]. Sampling from these communication modes would allow for finer scale look at the evolution of phototransduction genes.

As it stands, we have successfully identified several genes that are strong candidates for further positive selection studies. In the case of inaC, we have also identified a tantalizing area of study into the loss of certain phototransduction genes across the beetle tree of life. Further study will surely help our understanding of positive selection in terms of adaptation to differing sensory niches.

97 ACKNOWLEDGEMENTS

We would like to thank the members of the Bybee Lab at BYU as well as Jim Lloyd and

Marc Branham for access to specimens. We further thank members of the Bybee lab for assistance with Illumina library preparation We thank the BYU DNA Sequencing Center and the

Microarray and Genomic Analysis Core Facility at the Huntsman Cancer Institute at the

University of Utah for transcriptome sequencing as well as Perry Ridge, David Morris, Ryan

Hillary, and Perry Wood for assistance with transcriptome assembly and analyses. We also are appreciative to BYU for access to the Fulton Supercomputer, as well as the Oakley lab for access to and assistance with the PIA pipeline. GJM was supported in part by the BYU Graduate

Student Fellowship. GJM, AS, and SMB were supported by grant from the National Science

Foundation (SMB; IOS-1265714 and DEB-1655981) and the American Philosophical Society

(SMB). Neither BYU, the NSF, nor the APS played a role in the design of the study, analyses, interpretation of data and writing of the manuscript.

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107 CHAPTER 3 FIGURES

Figure 1: Diagram of the basic insect phototransduction pathway (modified from Macias-Munoz et al. 2019). Components in gray represent parts of the pathway not sampled. Components in orange were sampled but were recovered in too few taxa for selection analysis. All other components are colored to reflect relative omega values as reported via the PAML analysis. Dark blue indicates high relative omega values whereas lighter blue yellow indicates low relative omega values.

108 1.00

0.75

0.50 PP

0.25

0.00

arr2 cam_ gaq gprk1 inac inad inae ninaC norpA trpl gene

Figure 2: Distributions of the PP of positive selection across phototransduction genes. Violin plots represent estimated kernel density MCMC posterior probability of positive selection (blue dots).

109 Chapter 4: Building appreciation for Natural History Collections from biological collection data.

Gavin J. Martinab*, Jamie L. Jensena, Connor McDonalda, Brandon Cunninghamb, Seth M. Bybeeab aDepartment of Biology, 4102 LSB, Brigham Young University, Provo, UT 84602, USA bMonte L. Bean Science Museum, Brigham Young University, Provo, UT 84602, USA

*Corresponding author: [email protected]

Key words: specimens, label data, fireflies

ABSTRACT

Natural history collections are currently underfunded and understaffed. There is a growing need to educate the general public on the importance of natural history collections for diverse scientific topics from species discovery to effects of climate change. Here we present on online module aimed at assessing differences in attitudes towards natural history collections as well as the effect on pro-environmental thinking in a group of religious, undergraduate, non- biology major students. We show statistical differences post-intervention suggesting that this module is effective at raising students ranking of importance of natural history collections to both scientific research and the general public.

110 INTRODUCTION

There is a growing and documented need for inquiry-driven, active science education

(NRC, 2003; Brewer and Smith, 2011; Cook et al., 2014). Rutherford (1990) stated: “Science, mathematics, and technology do not create curiosity. They accept it, foster it, incorporate it, reward it, and discipline it–and so does good science teaching…” (Rutherford, 1990). With these two ideas in mind us as educators can ask ourselves the following questions: How was our curiosity fostered in the various “-ology” courses which incorporate specimens? How many of us make use of specimens in our research projects? How can we use specimens in our introductory level classrooms in hands on activities? Natural history collections are the historical repository against which we can measure our rapidly changing world. Changes in policy, land use, technology, population and especially climate mean that the world is more heavily impacted now than ever before. Natural history collections provide the information we need to understand the responses of organisms to these changes (Suarez and Tsutsui, 2004; Winker, 2004; Wandeler et al., 2007). Further, they allow us to harness these collections for genetic studies (for example

Neanderthal genomics: Prüfer et al 2014) to not only understand the history of organisms around us but even our own as a species. There are approximately 2–4 billion specimens in natural history collections worldwide (Ariño, 2010). While the public side of natural history museums

(i.e., exhibits, activities, etc.) are broadly appreciated by the public, the non-public side of natural history museums, the collections and laboratories, are often as easily accessible and thus are not as fully understood and or appreciated by the general public (Suarez and Tsutsui, 2004). Further, natural history collections also represent a vast resource and network for the improvement of biological education. Increasing efficiency in the biology classroom can best be achieved when mirroring what is done in the laboratory, especially focusing on inquiry-driven science (Feldman

111 et al., 2012). Scientific teaching seeks to mirror the discovery process in the classroom

(Handelsman et al., 2004). Using collections and mirroring the research that happens in collections is an excellent way to increase inquiry-driven science for students while also building a more complete understanding for collections among students that represent the general public.

Specimens in Science:

Specimens in natural history collections are extremely valuable to scientific research. For example, natural history collections represent a fantastic resource for measuring/predicting changes in population distribution (Elith et al., 2006). Measuring population change/historical biogeography is usually not possible given a lack of historical data with which to compare current populations, this problem is alleviated via the use of natural history collections as a proxy, which for many taxa, contain specimens dating to tens and often hundreds of years

(Shaffer et al., 1998, Shaw et al., 2004, Vik et al., 2008, Jeppsson et al., 2010). Natural history collections are also a vital resource in identifying invasive species and even ways to control them

(Suarez and Tsutsui, 2008; Lister, 2011). This can be done by finding an invasive species historical distribution and locating potential biological control agents (e.g., predators, viruses, etc.) that have controlled the numbers of that species throughout its evolution, thus, providing a critical historical perspective (Crawford and Hoagland, 2009). Specimens from natural history collections are perhaps best known for their use in species discovery. For example, in a revision of the Australian beetle Deretaphrus, Lord and McHugh (2013) examined 1,900 specimens across 44 museums and discovered seven new species. In a similar study of Australian tardigrades, eleven new species were discovered (Claxton, 1998). This type of result is not

112 unusual, as most of the new species which have been described were described from specimens deposited in natural history collections (Green, 1998; Fontaine et al., 2012). Just last year, 412 new species were described from one natural history collection alone, including two dinosaurs

(Chapelle et al., 2019 & Maidment et al., 2019), an extinct bandicoot (Travouillon et al., 2019), several (possibly endangered) plants (ex. Gouvêa, 2019) and 12 polychaete worms (Wiklund et al, 2019).

The historic nature of natural history collections (e.g. series of specimens collected over large periods of time) allows for further comparative studies testing hypotheses, for example those associated with climate change and/or habitat loss. In a study of passerine birds in Israel,

Yom-Tov (2001) used natural history collections to measure several species for both body mass and tarsus length and found a trend of decreasing body size from the years 1950 to 1999, coinciding with an increase in average summer temperature of .26°C. Global warming is known to affect many biological aspects of an organism (Hughes, 2000), and as such represents a potential explanation for this trend. Similarly, in a study of stream-inhabiting beetles, it was shown that an increase in size, coupled with a change to a more streamlined body, had occurred during a 60-year period and that a decrease in mean annual temperature was the most predictive variable for this trend (Babin-Fenske et al., 2008). Such studies would be nearly impossible without natural history collections. Further, natural history collections function as the repository of specimens from such studies so study can continue into the future and the associated data from each specimen can be preserved.

Specimens in Scientific Teaching:

113 Specimen-based learning has the potential to be an incredible tool in formal undergraduate education. Specimens bring biology into the classroom and help students appreciate that the concepts they are learning apply to living organisms. This can also help alleviate the problem of attrition due to many students acquiring the misconception that biology is all about memorization (Handelsman et al., 2007). Using specimens and specimen metadata can drive inquiry-based education (Cook et al., 2014), and teach students that biology is really about thinking logically and solving problems, and that discovery is at its core.

Traditionally exposure to biological specimens in formal university education settings is usually limited to upper-level “ology” type courses (e.g. entomology, botany, ornithology).

Students in these classrooms learn first-hand the beauty and excitement hidden in the natural world (Wallace, 1869). The problem arises in that these experiences are rarely transferred to introductory level biology courses (Powers et al., 2014) and even those experiences in upper- level “ology” courses rarely focus on the importance of natural history collections and the scientific data that can be harnessed from them.

Lacey et al. (2017) provided modules demonstrating how to incorporate natural history collection data to test hypotheses of adaptation to climate change. Several other studies focusing on formal science education have demonstrated the effectiveness of modules centered around local geography and culture (Castagno and Brayboy, 2008; Kisker et al., 2012; Barnhart, 2014;

Lipka et al., 2014). Adding to this research, Anderson et al. (2017) developed a series of modules centered around the collection at the University of Alaska Museum which provide an example of how to utilize local collections in the classroom.

These modules are a wonderful resource for educators who want to include specimen- based active learning in the classroom. From any of the above listed papers, with a little

114 modification in most cases a learning modules can quickly be assembled. For example, for assembling a module to teach biogeography and given the accessibility of metadata databases, it is fairly straightforward to download a dataset comprising all the county records for a given species. Beforehand, students can be given information about the species and asked to predict a species ideal environment. The dataset can then be incorporated into one of various ecological models so that students can analyze the data and conclude whether their hypothesis is supported or rejected.

Natural history collections provide an opportunity to use specimens to increase awareness of diversity as they provide a way to interact with biodiversity on a personal level (Efthim, 2006;

Kimble, 2014; Pickering et al. 2012; Monfils et al., 2017). As one of the more diverse groups of life, insects offer the greatest opportunity to teach students about biodiversity (Wilson, 1992;

Mora, 2011). When most students think of insects, they may only think of the most common varieties: butterflies, ants, bees, cockroaches, mosquitos. With more than one million described species, and perhaps as many as 30 million species total (Erwin, 1982), there is much more for students to learn about the world around them, and insects are a fantastic place to start this inquiry. Insects also make up the majority of the holdings in many of the world’s natural history collections (Holmes et al., 2016). This is due in part to the general ease of collecting insects.

Usually, only a hand net and an eye for the obscure is needed. In addition to this is the ease of collecting several specimens, sometimes several hundred at a time. The small size of the specimens makes storage, and more importantly transport, of thousands of specimens relatively easy. In addition to the physical specimens, and perhaps more important/useful in the classroom, is the data associated with each specimen such as the label. Label data will ideally contain general locality information, and for more recently collected specimens specific GPS

115 coordinates. Labels should also reference the date of collection, the collector as well as any special collecting techniques or host plant information.

This metadata associated with each specimen from natural history collections is perhaps the most meaningful to educators. In many instances, use of actual specimens is too difficult.

Travel to a collection can take too long, and loan of necessary specimens can exceed the ability of local collection managers. In these cases, the metadata can still be used. For example, in several databases, housed across the world, there are thousands of data points that instructors can use in bringing the scientific process into the classroom. See for example the databases iDigBio

(https://www.idigbio.org/) and SCAN (https://scan-bugs.org/portal/). Through these databases, anyone can download datasets containing the records of hundreds of firefly sightings recorded in several major natural history collections across the United States. Included in this data usually are observations of locality, date, time of day, and in many cases, other useful information such as type of vegetation and/or habitat. This information can be curated and brought into the undergraduate classroom to investigate patterns of firefly distribution. Students can be challenged to develop hypotheses that would explain the data, and to draw predictions from these hypotheses. Other, more locally-focused, independent databases (i.e. https://nhmu.utah.edu/fireflies, based largely on museum specimens) can then be consulted.

Using such resources, students can explore the scientific method in a meaningful, personalized way by testing predictions and revising hypotheses. Utilizing the metadata with these collections can bring scientific inquiry into the classroom in a way that cannot be done otherwise.

Following this line of thinking we seek to add to the current literature an online learning module with two objectives, both aimed at undergraduate students in non-majors introductory biology courses. The first of these is to increase student perception of the importance of natural

116 history collections. Our second objective is to increase pro-environmental thinking as assessed by the Revised-New Environmental Paradigm scale (Dunlap and Van Liere, 1978, Dunlap et al.,

2000). As stated above, a current major line of study within natural history collections is that of climate change, but other issues can also be explored using museum metadata such as changes in species distribution due to such factors as environmental degradation and/or pollution.

117 MATERIALS AND METHODS

Undergraduate students in two introductory biology courses for non-majors were selected for this study (~350 total). Students were administered a pre-assessment composed of the 15 R-

NEP scale questions with two additional likert-scale questions: 1: Natural history collections are important for scientific research and 2: Natural history collections are important for the general public. The student then participated in a short online module (described in detail below) followed by a post-assessment. The post assessment was comprised of the same 17 questions above; however, we added several questions after seeing the responses to the pre-assessment.

Two free-response questions were added to ascertain more detail about specific responses: 1:

Concerning statement 12, how do you define “rule”? and 2: Concerning statements 16 and 17, please define a “Natural history collection” to the best of your understanding. Because this module centered on fireflies in the Utah region, four more questions were added to investigate if our choice of organism had an effect: 1: On a scale of 1 (no concern) to 10 (extreme concern) how concerned are you with the extinction of fireflies? 2) On a scale of 1 (no concern) to 10

(extreme concern) how concerned are you with the extinction of pandas? 3) On a scale of 1 (no concern) to 10 (extreme concern) how concerned are you with the extinction of moss? and 4) In your opinion, how would you rank the above three organisms (fireflies, pandas, and moss) in the importance of preventing them from going extinct? The 17 likert-scale questions were compared pre- and post- using a paired sample T test implemented in SPSS. Additionally, the 15 R-NEP scale questions were divided into six groups, the total score as well as the five facets of an ecological worldview (Dunlap et al. 2000): the reality of limits to growth, antianthropocentrism, the fragility of nature’s balance, rejection of exemptionalism, and the possibility of an ecocrisis.

Module

118 The online module was designed with specimen data from the Monte L. Bean Science

Museum (MLBM) at Brigham Young University. Fireflies were chosen as the module organism due to several factors: fireflies are the main area of expertise of two of the authors (GJM &

SMB), they are a gender-neutral insect (i.e., they are equally attractive to or disliked by both female and male students), fireflies are charismatic, well known animals rarely encountered in

Utah, and several populations under study by the authors are at risk of extirpation or have already been extirpated.

The module begins with a brief description of the MLBM and the primary purpose of its natural history collections (i.e. to advance scientific knowledge, address societal issues, and to increase scientific literacy). Students are instructed about the major holdings of the MLBM and taught that one of the major strengths of the MLBM is the insect collection with over 2 million specimens. Students are then given a brief background on fireflies with facts such as: fireflies are beetles, many species rely on bioluminescence (use of light for communication) for sexual reproduction, and that because of this, fireflies are highly susceptible to light pollution.

Students are then given an excel spreadsheet of curated data from MLBM specimens and told to examine the data to determine where in Utah and at what time of year fireflies have traditionally been found. Students are then given instructions on how to use the website simplemappr.net to achieve these goals. Students were then given a short, formative assessment to determine if they could correctly interpret the data. This assessment consisted of one multiple choice question:

If you wanted to go and see fireflies in Utah, where and when would you go?

A. Lake Bottom Canal, Provo; last week of June

119 B. Goshen Marshes; First week of June

C. Spring Lake, Payson; Last week of February

D. Dugway; First week of July.

All location responses except Dugway were listed on the excel file, and all times of year responses except February were also included in the excel file. This was designed to lead students to select either A. or B. If students choose A., then they are congratulated on selecting a correct answer. However, students are then instructed that although fireflies have been encountered at this location before, fireflies have not been seen there for several years. Students are presented with images of what the location looks like now where they can observe that locality is now bordered by a freeway with several billboards and extensive lighting. Students are then asked to hypothesize why fireflies are no longer found at this locality. They are then directed to the slides for option B. If students originally choose option B. they are congratulated for choosing a correct answer and presented with a short youtube clip of fireflies in Utah. They are then directed to the slides for option A. After seeing both options, students are asked to compare the two habitats. Students are then asked to think about the unintended effects on nature of certain human needs (i.e. building a well-lit freeway for safety reasons that at the same time increases light pollution). After a series of thought questions and statements about how the only way we know certain populations existed is through natural history collections, students are left with the final question: When human development is at odds with the natural world, what, if any, is our responsibility to protect it?

120 RESULTS

The pre-assessment resulted in 326 responses, while the post-assessment had 294 responses. After curating the data for students who took both the pre- and post-assessments, we had a sample size of 267. Analysis of the responses from the 267 students showed no significant differences looking at the total score or any of the five facets of the R-NEP with the exception of the facet “possibility of an ecocrises” (P=.049). When looking at individual items, there was a significant difference (P< .05) for the following items: question 3 (P=.012), question 5 (P=.005), and question 7 (P=.032). However, it should be noted that none of these questions showed any practical difference between pre- and post-assessments. Where we saw the highest differences were in the additional questions 16 (P<.001) and 17 (P<.001). Not only did we see statistically significant changes in these two items, we also saw practical changes, especially in the composition of answers (figs. 1 and 2). Pre-intervention for question 16 (Natural history collections are important for scientific research), 83 students strongly agreed, 126 somewhat agreed, 52 neither agreed nor disagreed, one somewhat disagreed, and three strongly disagreed.

Post-intervention, 129 students strongly agreed, while only 98 somewhat agreed, and 27 neither agreed nor disagreed, while seven somewhat disagreed, and three strongly disagreed. This pattern was the same for question 17 (Natural history collections are important for the general public). Pre-intervention 67 students answered strongly agree (117 somewhat agree and 71 neither agree nor disagree, six somewhat disagreed and four strongly disagreed). Post- intervention 109 students answered strongly agree (98 somewhat agree and 46 neither agree nor disagree, eight somewhat disagreed and three strongly disagreed).

121 DISCUSSION

While we did see gains in the number of students agreeing with the importance of natural history collections to both science and the general public, we did not see the gains we expected in pro-environmental thinking as assessed by the R-NEP. There are several possible factors that can explain this. A large portion of the online module was dedicated to explaining what natural history collections are. Perhaps the animal biology portion did not make a clear enough case that fireflies in Utah are at critical risk of extinction. It is also possible that the example organism

(fireflies) are not seen as a vital component of the ecosystem. At the end of the post-assessment students were asked to rank various organisms. However, the data seem to tell two different stories demonstrating that our students may care less about fireflies than we previously thought.

When asked to quantify the level of concern for each organism pandas receiving the highest support (average: 6.56), then moss (5.88), leaving fireflies (5.58) with the lowest level of concern. This pattern was surprising to us. Students expanded their reasoning in the free response section where they were asked to rank the three organisms. Many students placed moss as the most important organism to save (based on their stated assumption that mosses are more important to the ecosystem) and fireflies as the least important. It would be interesting if more/different examples of this pattern of extirpation in Utah could affect more of a change in the R-NEP. It is also possible that utilizing specimens from a more global organism could affect a desired change.

While no gain was shown in pro-environmental thinking, we did see substantial gains in the “importance” students placed on natural history collections. What is additionally encouraging is the fact that our students started relatively high in agreeance (table 1). It is also worth noting that while we did not see a large practical change in the average level of agreement (shift in

122 average for question 16 of 0.215 and question 17 of 0.242), we did see a major shift in the composition of each response group. For example, in question 16 students were asked their agreement with the statement “Natural history collections are important for scientific research.”

At the beginning of the study the majority of the students answered either “somewhat agree” or

“neither agree nor disagree.” Post-intervention both of these response groups were diminished with a marked increase in the number of students answering “strongly agree” (Fig. 1). This same pattern was shown in question 17 “Natural history collections are important for the general public.” Although the general pattern in gains was the same between both questions, we did see a smaller component of the students who strongly agreed with question 17 as opposed to those who strongly agreed with question 16 (129 students post-intervention in question 16 vs.

109 students post-intervention in question 17).

123 CONCLUSIONS

We’ve argued for the value of using data from natural history collections in the classroom as a means to increase student appreciation for both collections and the environment. As stated above, the diversity of life preserved in natural history collections is used in various scientific pursuits and inherent value of natural history collections cannot be quantified. Not only are collections used in the discovery of life (a fact the general public might not be aware of), these collections are also actively being used in a wide variety of studies that are important to human origins and existence as well general earth health, including the maintenance and protection of biodiversity in today's rapidly changing world. If we can affect even a small change in attitudes towards the importance of these collections, then as biologists we have the responsibility to do what we can to ensure their future protection.

124 ACKNOWLEDGEMENTS

The authors are extremely grateful to Dr. Michael Whiting (curator) and Dr. Shawn Clark

(collections manager) at the Monte L. Bean Science Museum for access to the firefly specimens from which data were gathered. We also thank Laura Sutherland (BYU) for assistance with

SPSS.

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131 CHAPTER 4 TABLES

Table 1: Summary statistics of paired differences for paired samples test

132 CHAPTER 4 FIGURES

Figure 1: Scaled bar graph of responses for question 16.

Figure 2: Scaled bar graph of responses for question 17.

133 APPENDIX

Supplemental material for chapter 1

Supp. Table 1. Genomic resources used to develop Anchored Hybrid Enrichment probe kit design.

Species Source Type Source Pyractomena borealis Genome Assembled Stanger-Hall Lab UGA Phausis reticulata Genome Assembled Stanger-Hall Lab UGA Photinus pyralis Genome Assembled Stanger-Hall Lab UGA Photinus scintillans Genome Assembled Stanger-Hall Lab UGA Photinus australis Transcriptome Assembled Stanger-Hall Lab UGA Pyractomena Transcriptome Assembled Stanger-Hall Lab UGA Photinus carolinus Transcriptome Assembled Stanger-Hall Lab UGA Pyractomen borealis Transcriptome Assembled Stanger-Hall Lab UGA Lucidota atra Transcriptome Assembled Stanger-Hall Lab UGA Photuris sp. Transcriptome Assembled Stanger-Hall Lab UGA Photuris frontalis Transcriptome Assembled Stanger-Hall Lab UGA Photinus scintillans Transcriptome Assembled Stanger-Hall Lab UGA Cantharidae Transcriptome Assembled Stanger-Hall Lab UGA Phausis sp. Transcriptome Assembled Stanger-Hall Lab UGA Photinus macdermotti Transcriptome Assembled Stanger-Hall Lab UGA Photinus pyralis Transcriptome Assembled Stanger-Hall Lab UGA Collected by Center for Anchored Photinus pyralis Genome Unassembled Phylogeny Collected by Center for Anchored Photinus borealis Genome Unassembled Phylogeny Collected by Center for Anchored Phausis reticulata Genome Unassembled Phylogeny

134 Supp. Table 2. Summary data for probe design and assembly.

CenterID SampleID Family Species ID Source nRawReads nRawBases nMergedReads nMergedBases nUnmergedReads nUnmergedBases nLoci125 nLoci250 nLoci500 nLoci1000 AvgLocLenAll %OnTarget ReadsPerLocus AvgNHomologs DupRateAll I16321 CO1077 Elateridae Pyrophorous_sp. AHE 8054558 1523955600 2974706 625114916 2105146 631543800 495 493 479 317 1363.005814 14.03769892 2190.25969 1.143410853 0.199055593 I16323 CO1517 Lampyridae Pteroptyx_sp. AHE 8266388 1600902600 2930046 651209787 2406296 721888800 507 505 493 377 1514.943798 14.24289012 2408.532946 1.218992248 0.19489652 I16324 CO1544 Phengodidae Phengodes_sp. AHE 6835086 1295024100 2518339 553028189 1798408 539522400 425 405 298 92 759.5872093 1.928324895 230.1647287 1.031007752 0.109952101 I16325 CO1545 Lycidae Calopteron_reticulatum AHE 5502387 1039839600 2036255 443879308 1429877 428963100 399 372 230 37 607.4399225 1.410363874 134.2248062 0.974806202 0.107284101 I16326 CO1546 Lampyridae Photuris_congener AHE 5615974 1006783200 2260030 466680626 1095914 328774200 514 514 510 362 1461.841085 15.01289855 1303.062016 1.290697674 0.130414625 I16327 CO1547 Lampyridae Photinus_pyralis AHE 16499528 3059598300 6300867 1312551027 3897794 1169338200 515 515 515 502 2134.98062 29.27987011 9162.331395 1.408914729 0.344327095 I16328 CO1548 Cantharidae Cantharidae_sp._1 AHE 7703540 1323378300 3292279 664489646 1118982 335694600 446 423 332 88 816.3042636 4.301918189 473.1550388 1.418604651 0.116874604 I16329 CO1549 Lampyridae Phausis_reticulata AHE 15118389 2653081200 6274785 1301607652 2568819 770645700 516 516 515 441 1704.988372 20.37712612 5005.399225 1.441860465 0.215415375 I16330 CO1550 Lampyridae Pterotus_obscuripennis AHE 2631769 477269400 1040871 195705449 550027 165008100 499 494 468 259 1206.563953 16.04980882 662.8197674 1.187984496 0.135697688 I16331 CO1551 Lampyridae Lucidota_atra AHE 13975241 2719955100 4908724 1083782931 4157793 1247337900 514 514 513 489 2051.20155 22.48515881 6564.600775 1.42248062 0.279362346 I16332 CO1552 Lampyridae Pyropyga_decipiens AHE 6841020 1207601700 2815681 579236362 1209658 362897400 513 511 502 388 1538.176357 22.94233035 2504.689922 1.248062016 0.17970001 I17942 CO1049 Lampyridae Photuris_divisia AHE 2762668 613009800 719302 139617219 1324064 397219200 509 492 415 190 1068.218992 9.441603663 612.627907 1.437984496 0.203089106 I17943 CO1057 Lampyridae Photinus_stellaris AHE 11879850 1855642200 5694376 965809152 491098 147329400 515 515 510 350 1426.45155 18.12248194 2283.412791 1.465116279 0.257577155 I17944 CO1060 Lampyridae Lamprocera_sp._1 AHE 3418001 726653100 995824 214207556 1426353 427905900 509 509 505 406 1566.418605 15.16945239 1220.139535 1.118217054 0.171310888 I17945 CO1062 Lampyridae Pyrogaster_sp. AHE 3215997 695246400 898509 176093905 1418979 425693700 503 494 440 251 1186.304264 6.320652284 461.9457364 1.164728682 0.152240302 I17946 CO1063 Lampyridae Pyractonema_sp._1 AHE 4998969 1091714100 1359922 281202836 2279125 683737500 513 513 508 461 1788.007752 19.30368563 2382.821705 1.153100775 0.204650984 I17947 CO1067 Lampyridae Heterophotinus_sp. AHE 5489288 1074434100 1907841 397754708 1673606 502081800 514 513 511 417 1614.525194 17.42527166 1934.850775 1.253875969 0.191417075 I17948 CO1069 Lampyridae Aspisoma_sp. AHE 5238292 1069047600 1674800 324784006 1888692 566607600 512 507 476 256 1218.257752 5.382058758 567.7751938 1.339147287 0.146498978 I17949 CO1071 Lampyridae Lampyris_noctiluca AHE 2781293 628754700 685444 140099603 1410405 423121500 510 509 492 333 1392.463178 11.88849293 827.1375969 1.222868217 0.191943515 I17950 CO1074 Lampyridae Photinus_macdermotti_1 AHE 2454648 579877800 521722 108257444 1411204 423361200 515 515 515 479 1804.344961 36.81186659 2496.205426 1.141472868 0.376920482 I17951 CO1080 Lampyridae Pyropyga_nigricans AHE 1503881 344211000 356511 70341962 790859 237257700 503 494 449 242 1172.145349 19.5668648 741.4709302 1.26744186 0.251480827 I17953 CO1092 Lampyridae Microphotus_sp. AHE 3887432 816897900 1164439 239275730 1558554 467566200 513 510 500 368 1427.945736 12.9983889 1125.416667 1.251937984 0.187647302 I17955 CO1197 Lampyridae Ellychnia_sp. AHE 4012664 890438100 1044537 217271944 1923590 577077000 515 515 511 418 1586.781008 29.6462319 2958.434109 1.304263566 0.393124639 I17956 CO1457 Lampyridae Lamprocera_sp._3 AHE 2050178 468691500 487873 100593125 1074432 322329600 511 508 493 360 1455.540698 19.94309386 1053.53876 1.166666667 0.242661133 I17957 CO1505 Lampyridae Luciolinae_nr._Luciola_1 AHE 3281385 676734300 1025604 197665978 1230177 369053100 465 454 394 172 991.1550388 6.118087487 417.5426357 1.15503876 0.177216795 I17958 CO1509 Lampyridae Luciolinae_nr._Luciola_4 AHE 6022276 1203783900 2009663 383649903 2002950 600885000 473 464 417 239 1124.781008 4.929365414 599.1686047 1.137596899 0.138258378 I17959 CO1510 Lampyridae Luciolinae_nr._Luciola_3 AHE 1321637 305284800 304021 61326337 713595 214078500 471 450 393 190 1017.899225 11.84681952 404.2655039 1.186046512 0.159270137 I17960 CO1520 Lampyridae Australoluciola_nigra AHE 1548376 340276500 414121 81928449 720134 216040200 469 447 391 196 1017.521318 9.962058794 365.4631783 1.122093023 0.154549371 I17961 CO1524 Lampyridae Diaphanes_sp._3 AHE 10236305 1823219100 4158908 713584600 1918489 575546700 511 510 502 393 1539.922481 7.263801057 1161.23062 1.226744186 0.145501742 I17962 CO_ARG Lampyridae Pyractonema_sp._2 AHE 1448649 343927800 302223 58794417 844203 253260900 505 485 408 201 1064.348837 11.45137322 441.7344961 1.323643411 0.181546278 I22180 CO1281 Lampyridae Pollaclasis_bifaria AHE 5361875 964081200 2148271 464287389 1065333 319599900 485 474 372 122 916.125969 4.037271358 332.9922481 1.220930233 0.184810218 I22181 CO1285 Phengodidae Zarhipis_integripennis AHE 6966354 1423295700 2222035 483834131 2522284 756685200 434 424 329 113 819.2732558 2.728061075 397.8236434 1.07751938 0.16768182 I22182 CO1286 Telegeusidae Telegeusis_nubifer AHE 11079449 2156628000 3890689 852226624 3298071 989421300 433 422 340 100 853.370155 2.978015683 640.9399225 1.207364341 0.185668472 I22183 CO1287 Lampyridae Lamprohiza_splendidula AHE 2582866 476638500 994071 221546315 594724 178417200 434 365 199 61 773.1569767 3.200904462 132.503876 1.19379845 0.189929075 I22184 CO1290 Lampyridae Bicellonycha_sp._1 AHE 4358236 756701100 1835899 365242624 686438 205931400 495 489 403 147 998.127907 6.037259496 361.0988372 1.333333333 0.197361462 I22185 CO1291 Lampyridae Abscondita_cerata AHE 11270679 2038119600 4476947 976316041 2316785 695035500 509 506 499 337 1415.534884 15.70973554 3137.846899 1.23255814 0.276472451 I22186 CO1292 Lampyridae Diaphanes_pectinealis AHE 2935214 514502100 1220207 261187421 494800 148440000 512 508 461 212 1109.282946 15.91351156 645.7887597 1.387596899 0.226779217 I22187 CO1293 Lampyridae Pyrocoelia_pygidialis AHE 8520795 1610228100 3153368 704774420 2214059 664217700 515 514 507 369 1493.172481 14.68195208 2399.864341 1.259689922 0.223381016 I22188 CO1294 Lampyridae Lamprigera_yunnana AHE 6296186 1177186800 2372230 529356084 1551726 465517800 483 470 417 147 984.0387597 6.035077206 666.5232558 1.294573643 0.186699719 I22189 CO1295 Lampyridae Emeia_pseudosauteri AHE 7810438 1392658800 3168242 677675872 1473954 442186200 509 504 475 272 1275.362403 12.40351785 1600.156977 1.271317829 0.216633858 I22190 CO1296 Lampyridae Pygoluciola_qinqyu AHE 6984522 1212319200 2943458 624965824 1097606 329281800 503 496 465 257 1234.21124 12.06669895 1270.468992 1.180232558 0.206473689 I22191 CO1297 Lampyridae Asymmetrica_circumdata AHE 9628812 1567447200 4403988 888328676 820836 246250800 499 492 413 164 1018.486434 7.268208772 833.2383721 1.277131783 0.215088435 I22192 CO1298 Lampyridae Curtos_bilineatus AHE 6970579 1400747400 2301421 527529376 2367737 710321100 499 494 476 260 1240.215116 9.128610861 1399.255814 1.228682171 0.22595973 I22193 CO1299 Lampyridae Vesta_saturnalis AHE 3669322 568657800 1773796 312389624 121730 36519000 479 450 305 95 837.0406977 5.331429698 182.2829457 1.217054264 0.201562212 I22195 CO1302 Lampyridae Stenocladius_shirakii AHE 4798125 854765400 1948907 361108530 900311 270093300 480 470 414 145 981.7364341 10.13888537 752.001938 1.246124031 0.231223539 I22196 CO1303 Lampyridae Curtos_obsuricolor AHE 5254327 988770600 1958425 438398559 1337477 401243100 497 488 437 202 1119.007752 8.637329416 818.8372093 1.230620155 0.147356144 I22197 CO1304 Rhagophthalmidae Rhagophthalmus_ohbai AHE 10709528 2046194400 3888880 753159973 2931768 879530400 490 481 444 216 1125.434109 5.116707269 1023.156977 1.189922481 0.171806533 I22198 CO1305 Lampyridae Vesta_impressicollis AHE 14862014 2638708200 6066320 993150286 2729374 818812200 507 505 491 299 1296.916667 3.406265167 740.6996124 1.236434109 0.154413002 I22199 CO1306 Lampyridae Tenaspis_angularis AHE 5501152 934367400 2386594 493125938 727964 218389200 513 512 500 322 1374.666667 22.04979987 1712.197674 1.187984496 0.168724503 I22200 CO1307 Lampyridae Pleotomodes_needhami AHE 5078790 921195300 2008139 436447141 1062512 318753600 514 513 504 317 1367.505814 14.84173623 1233.668605 1.230620155 0.143418553 I22201 CO1308 Lampyridae Drilaster_sp. AHE 2737977 524438100 989850 218162994 758277 227483100 492 482 407 163 1005.637597 9.227340159 459.4050388 1.162790698 0.136382968 I22202 CO1309 Eucnemidae Microrhagus_pygmaeus AHE 4622214 894041100 1642077 353645000 1338060 401418000 457 436 380 138 961.3023256 9.628312737 878.620155 1.21124031 0.178724342 I22203 CO1310 Elateridae Ampedus_pomonae AHE 3100225 583043700 1156746 254227565 786733 236019900 469 455 359 94 853.5465116 7.028300085 376.5503876 1.213178295 0.162167943 I22204 CO1245 Lampyridae Psilocladus_sp._1 AHE 2661444 497095800 1004458 206256101 652528 195758400 486 477 403 152 979.9767442 8.253322683 370.7674419 1.414728682 0.129574769 I22205 CO1255 Lampyridae Curtos_sp. AHE 4597612 992756100 1288425 294364504 2020762 606228600 499 493 474 296 1301.742248 14.84799622 1705.75969 1.143410853 0.182510085 I22206 CO1459 Lampyridae Lamprocera_sp._2 AHE 5066172 985468500 1781277 400279517 1503618 451085400 513 512 508 384 1532.651163 18.11812443 1848.29845 1.17248062 0.179015856 I22207 CO1516 Lampyridae Diaphanes_sp._2 AHE 6589017 1269525300 2357266 535449919 1874485 562345500 515 514 505 368 1494.649225 15.43808993 2037.579457 1.226744186 0.180496136 I22208 CO1523 Lampyridae Diaphanes_sp._4 AHE 22109420 3507629100 10417323 1635635219 1274774 382432200 509 508 496 326 1370.339147 3.877531507 945.4806202 1.242248062 0.060898502 I22209 CO1525 Lampyridae Luciolinae_nr._Luciola_2 AHE 4596407 924579900 1514474 341305638 1567459 470237700 497 492 467 248 1205.982558 14.14094585 1430.879845 1.228682171 0.195748202 I22210 CO1528 Lampyridae Photinus_sp._2 AHE 4332493 845324400 1514745 333288494 1303003 390900900 515 515 513 359 1450.829457 19.95732376 1689.195736 1.381782946 0.175786029 I22212 CO1534 Lampyridae Luciolinae_sp. AHE 3846810 705762900 1494267 332212307 858276 257482800 500 498 454 211 1127.04845 18.61366979 1256.50969 1.218992248 0.191062561 I22213 CO1518 Lampyridae Luciola_sp._1 AHE 3846336 751294500 1342021 302297709 1162294 348688200 498 497 476 251 1219.372093 16.28227793 1288.718992 1.246124031 0.191878641 I22214 406 Lampyridae Photuris_frontalis AHE 1345861 250450500 511026 109173044 323809 97142700 512 511 498 312 1328.312016 30.47878353 686.2887597 1.271317829 0.168980195 I22215 4081 Lampyridae Pyractomena_borealis AHE 4704887 972074100 1464640 333070400 1775607 532682100 515 515 511 432 1674.618217 24.8098497 2695.131783 1.203488372 0.245132264 I22216 6091 Lampyridae Ellychnia_corrusca AHE 4960097 984591000 1678127 373143066 1603843 481152900 515 514 511 422 1614.050388 28.28874624 3013.664729 1.209302326 0.279058254 I22217 10061 Lampyridae Photinus_floridanus AHE 12891853 2510490000 4523553 1017813890 3844747 1153424100 515 515 514 468 1779.897287 20.69914713 5561.422481 1.501937984 0.279372298 I22218 1028 Lampyridae Photinus_australis AHE 3918557 793659300 1273026 282382839 1372505 411751500 515 515 515 425 1631.282946 20.38677012 1662.374031 1.209302326 0.199154573 I22219 12271 Cantharidae Cantharidae_sp._2 AHE 3948841 761508900 1410478 308017008 1127885 338365500 492 487 461 274 1251.062016 20.82682565 1646.021318 1.312015504 0.232467979 I22220 14611 Lampyridae Photinus_granulatus AHE 4689957 893500800 1711621 363920599 1266715 380014500 515 514 511 420 1610.071705 23.51519228 2046.30814 1.226744186 0.19619577 I22221 17651 Lampyridae Photinus_ardens AHE 6397015 1178635500 2468230 550042082 1460555 438166500 515 515 513 422 1625.455426 28.15865092 3247.560078 1.244186047 0.273195381 I22222 87151 Lampyridae Photinus_macdermotti_2 AHE 6593861 1260723600 2391449 528064163 1810963 543288900 515 515 512 449 1839.418605 28.79770009 3747.835271 1.238372093 0.286153066 I22223 8870 Lampyridae Photuris_quadrifulgens AHE 2270620 440786100 801333 179680862 667954 200386200 514 512 503 332 1362.416667 19.12378453 802.0310078 1.251937984 0.158143206 I22224 9013 Lampyridae Photinus_brimleyi AHE 3384853 697830300 1058752 239593983 1267349 380204700 515 515 514 427 1629.060078 25.50752613 1950.344961 1.197674419 0.199915181 I22227 9670 Lampyridae Photinus_carolinus AHE 6444470 1169490900 2546167 506725885 1352136 405640800 515 515 515 442 1667.056202 32.05034026 3626.203488 1.230620155 0.278001112 I23184 CO1211 Lampyridae Cladodes_illigeri AHE 6459158 1230070500 2358923 526267537 1741312 522393600 510 510 499 328 1378.620155 13.09478202 1601.300388 1.222868217 0.192594198 I23185 CO1212 Lampyridae Amydetes_fastigiata AHE 5746246 1025143500 2329101 500130829 1088044 326413200 502 496 443 222 1144.412791 9.805438205 866.5077519 1.244186047 0.171447521 I23186 CO1213 Lampyridae Ethra_axillaris AHE 9238023 1720136700 3504234 780360152 2229555 668866500 513 513 509 379 1513.203488 17.61750908 3076.589147 1.224806202 0.23158785 I23187 CO1214 Lampyridae Memoan_ciceroi AHE 5719120 1123818900 1973057 433330540 1773006 531901800 505 500 476 269 1246.732558 8.259348003 939.5503876 1.244186047 0.166277261 I23188 CO1215 Lampyridae Scissicauda_disjuncta AHE 12685974 2372630700 4777205 1041824336 3131564 939469200 515 514 513 419 1610.976744 18.6595879 4631.118217 1.300387597 0.246964294 I23189 CO1216 Lampyridae Psilocladus_sigillatus AHE 6017797 1146786900 2195174 459617511 1627449 488234700 505 498 469 221 1167.195736 7.465844377 834.9748062 1.281007752 0.176605544 I23190 CO1218 Lampyridae Psilocladus_sp._2 AHE 5350656 1010801100 1981319 396893766 1388018 416405400 501 497 469 263 1262.926357 12.9343857 1266.914729 1.259689922 0.192516573 I23191 CO1219 Lampyridae Vesta_sp. AHE 2803609 494330700 1155840 199713995 491929 147578700 475 449 340 113 894.9554264 4.615379831 179.5445736 1.143410853 0.195913787 I23192 CO1220 Lampyridae Lloydiella_uberia AHE 7629473 1432598400 2854145 621882690 1921183 576354900 497 492 465 239 1178.296512 14.95841238 2177.732558 1.234496124 0.265350248 I23193 CO1221 Lampyridae Atyphella_flammulans AHE 7115221 1396815900 2459168 554584666 2196885 659065500 500 497 474 260 1240.916667 11.25873409 1669.914729 1.261627907 0.198647516 I23194 CO1222 Lampyridae Australoluciola_sp. AHE 11643866 2188274700 4349617 866740495 2944632 883389600 507 501 491 312 1385.032946 17.68109547 3927.343023 2.098837209 0.27538219 I23195 CO1223 Lampyridae Diaphanes_sp._1 AHE 11413015 2305163400 3729137 793634167 3954741 1186422300 515 515 508 385 1542.063953 9.562529309 2353.678295 1.356589147 0.191529642 I23196 CO1224 Lampyridae Photinus_sp._1 AHE 13007394 2883871500 3394489 704738646 6218416 1865524800 516 515 514 472 1884.959302 26.57666346 8798.186047 1.217054264 0.31410159 I23197 CO1225 Lampyridae Luciola_sp._2 AHE 10957026 1802081400 4950088 1028745339 1056850 317055000 510 509 473 222 1176.527132 14.00399328 2012.73062 1.246124031 0.176065566 I23198 CO1226 Lampyridae Lychnacris_sp. AHE 8222419 1462671600 3346847 660287532 1528725 458617500 513 513 505 347 1402.949612 13.55031246 1685.143411 1.248062016 0.157490833 I23199 CO1227 Lampyridae Petalacmis_sp. AHE 8216532 1463563800 3337986 700491745 1540560 462168000 510 507 488 295 1305.027132 7.302289373 910.124031 1.187984496 0.127397204 I23200 CO1228 Lampyridae Bicellonycha_sp._2 AHE 5901870 1205289900 1884237 428139637 2133396 640018800 506 501 451 218 1123.172481 4.305613045 521.4108527 1.28875969 0.104098749 I23201 CO1229 Lampyridae Trisinuata_sp. AHE 10770867 2081610900 3832164 870457008 3106539 931961700 509 506 498 357 1523.556202 19.39862671 4423.517442 1.248062016 0.25968879 I23202 CO1230 Lampyridae Photinini_sp._ AHE 7229491 1349058300 2732630 554583816 1764231 529269300 514 514 514 375 1508.817829 16.04953028 2014.736434 1.550387597 0.135215269 I23203 CO1231 Lampyridae Lamprocera_sp._4 AHE 11097420 1982146800 4490264 962184654 2116892 635067600 515 514 512 380 1553.901163 14.93094065 2747.432171 1.182170543 0.178979764 I23204 CO1232 Lampyridae Lucio_blattinum AHE 7684151 1364858700 3134622 665150132 1414907 424472100 513 512 501 353 1414.583333 11.10201788 1317.306202 1.203488372 0.139531554 I23205 CO1233 Lampyridae Aspisoma_sticticum AHE 6928547 1273369200 2683983 572688179 1560581 468174300 514 514 510 372 1475.97093 19.34912748 2397.187984 1.199612403 0.199760077 I23206 CO1234 Lampyridae Cratomorphus_sp. AHE 11341325 2368855500 3445140 784234815 4451045 1335313500 514 514 512 399 1546.292636 8.165748748 2160.153101 1.253875969 0.143030634 I23207 CO1289 Lampyridae Phosphaenopterus_metzneri AHE 6625270 1236119400 2504872 521364115 1615526 484657800 513 513 512 361 1425.523256 16.28006901 1840.709302 1.63372093 0.142280856

135 Telegeusis nubifer

Microrhagus pygmaeus

Cantharidae sp. 1 100 Cantharidae sp. 2

Calopteron reticulatum 100 Pyrophorus sp. 100

100 Ampedus pomonae Rhagophthalmus ohbai 8 4 100 Phengodes sp. 100 Zarhipis integripennis

Stenocladius shirakii 100 Drilaster sp. 7 7 Pterotus obscuripennis 9 9 Pollaclasis bifaria

Lamprigera yunnana 100 100 Luciolinae nr. Luciola 1 100 Luciolinae nr. Luciola 2

Asymmetrica circumdata 100 100 Luciolinae sp. 100 Lloydiella uberia 100 Luciola sp. 1 100 7 7 Atyphella flammulans

100 Curtos bilineatus 100 Curtos obsuricolor 100 Australoluciola sp. 100 Australoluciola nigra 100 Pteroptyx sp. 100 100 Trisinuata sp.

Abscondita cerata 100 Luciola sp. 2 100 8 4 Luciolinae nr. Luciola 3 8 5 Emeia pseudosauteri 8 5 Pygoluciola qinqyu 100 Luciolinae nr. Luciola 4 100 Curtos sp.

Phausis reticulata 100 Lamprohiza splendidula

Amydetes fastigiata 100 Memoan ciceroi

Photuris quadrifulgens 100 100 Photuris divisia 100 100 Photuris congener 100 100 Photuris frontalis

Bicellonycha sp. 1 100 Bicellonycha sp. 2 100 Pyrogaster sp. 100 Vesta saturnalis 100 100 Vesta impressicollis 7 0 Vesta sp.

Psilocladus sp. 1 100 Psilocladus sigillatus 100 Psilocladus sp. 2

Cladodes illigeri

Tenaspis angularis 100

8 9 Lychnacris sp. Petalacmis sp. 100 Lucio blattinum

100 Lamprocera sp. 1 100 Lamprocera sp. 2 100 100 100 Lamprocera sp. 3 100 Lamprocera sp. 4

Cratomorphus sp. 100 Pleotomodes needhami 100 Microphotus sp. 100 Diaphanes pectinealis 100 100 Pyrocoelia pygidialis 100 Lampyris noctiluca 100 Diaphanes sp. 1 100 Diaphanes sp. 2 100 Diaphanes sp. 4 100 Diaphanes sp. 3

Pyractomena borealis 100 Aspisoma sp. 100 Aspisoma sticticum

100 Phosphaenopterus metzneri 100 Lucidota atra 100 Photinini sp. 100 Ethra axillaris

Heterophotinus sp. 100 Luciolinae Scissicauda disjuncta 100 100 Pyropyga decipiens Pterotinae 100 100 Pyropyga nigricans Ototretinae 100 Pyractonema sp. 1 100 Pyractonema sp. 2

Lamprohizinae Photinus sp. 1

Photinus sp. 2 Psilocladinae 100 Photinus floridanus 100 Amydetinae 100 Photinus macdermotti 2 100 Photinus stellaris Photurinae 100 Photinus ardens 100 100 Lampyrinae Photinus carolinus Photinus pyralis

incertae sedis 100 Ellychnia sp. 100 Ellychnia corrusca 100 Photinus macdermotti 1 100 Photinus granulatus 100 0.1 Photinus australis 100 Photinus brimleyi Supp. Fig. 1. Maximum Likelihood reconstruction of trimmed dataset aligned in MAFFT; log- likelihood = -10921284.680. Star at node depicts Lampyridae. Numbers at nodes indicate UFboot values. Unlabeled nodes are 100%.

136

Supp. Fig. 2. Coalescent reconstruction (ASTRAL) of full, untrimmed dataset aligned in MAFFT. Star at node depicts Lampyridae. Numbers at nodes indicate localized posterior probability.

137 Telegeusis nubifer Microrhagus pygmaeus 0.41 Cantharidae sp. 2 1 Cantharidae sp. 1 Calopteron reticulatum Ampedus pomonae 1 Pyrophorus sp. 1 Rhagophthalmus ohbai 1 0.57 Zarhipis integripennis 1 Phengodes sp. Stenocladius shirakii 0.73 Drilaster sp. 1 Pterotus obscuripennis Lamprigera yunnana 1 Luciolinae nr. Luciola 2 1 1 Luciolinae nr. Luciola 1 Asymmetrica circumdata 1 1 Luciolinae sp. 1 Lloydiella uberia 0.99 Luciola sp. 1 0.75 1 Atyphella flammulans 1 Abscondita cerata 1 Luciola sp. 2 Luciolinae nr. Luciola 3 0.8 Emeia pseudosauteri 1 1 Pygoluciola qinqyu 1 Curtos sp. 1 Luciolinae nr. Luciola 4 0.85 1 Curtos bilineatus 1 Curtos obsuricolor 1 Pteroptyx sp. 1 Trisinuata sp. 1 Australoluciola nigra 0.89 Australoluciola sp. Pollaclasis bifaria Lamprohiza splendidula 1 Phausis reticulata Psilocladus sp. 1 1 Psilocladus sigillatus 0.86 1 Psilocladus sp. 2 Amydetes fastigiata 0.66 1 Memoan ciceroi Photuris quadrifulgens 1 1 1 Photuris divisia 1 Photuris frontalis 1 1 Photuris congener Bicellonycha sp. 1 1 Bicellonycha sp. 2 0.77 Pyrogaster sp. 1 Vesta impressicollis 1 1 Vesta saturnalis 1 Vesta sp. Cladodes illigeri Tenaspis angularis 1 Lychnacris sp. Petalacmis sp. 1 1 Lucio blattinum Lamprocera sp. 2 1 1 Lamprocera sp. 1 1 1 0.84 Lamprocera sp. 3 1 Lamprocera sp. 4 Cratomorphus sp. 1 Pleotomodes needhami 1 Microphotus sp. Diaphanes pectinealis 1 1 Pyrocoelia pygidialis 1 1 Lampyris noctiluca 1 Diaphanes sp. 1 1 Diaphanes sp. 2 1 Diaphanes sp. 4 1 Diaphanes sp. 3 Pyractomena borealis 1 Aspisoma sp. 1 Aspisoma sticticum Phosphaenopterus sp. 1 1 Lucidota atra 1 Photinini sp. 1 Ethra axillaris Heterophotinus sp. 0.9 Scissicauda disjuncta 0.93 Pyropyga decipiens 1 1 Pyropyga nigricans Luciolinae 1 Pyractonema sp. 1 1 1 Pterotinae Pyractonema sp. 2 Photinus sp. 1 Ototretinae Photinus sp. 2 1 Photinus floridanus Lamprohizinae 0.98 Photinus macdermotti 2 1 1 Psilocladinae Photinus stellaris 1 Photinus ardens 1 Amydetinae 1 Photinus carolinus Photurinae Photinus pyralis 0.82 Ellychnia corrusca 1 Ellychnia sp. Lampyrinae 1 Photinus macdermotti 1 1 incertae sedis Photinus granulatus 0.97 Photinus australis 1 Photinus brimleyi

3.0 Supp. Fig. 3. Coalescent reconstruction (ASTRAL) of trimmed dataset aligned in MAFFT. Star at node depicts Lampyridae. Numbers at nodes indicate localized posterior probability.

138 Supplemental material for chapter 3

Supp. Table 1: Assembly summary statistics for transcriptomes new to this study

(Code=CO_###) as well as for those previously published.

139 Supp. Table 2: Presence absence data genes sampled in the phototransduction pathway. Gray box indicates presence. Orange box indicates presence of duplication in Photinus carolinus only.

140 Supp. Table 3: Summary of positive selection analyses carried out in PAML and BAli-Phy. P value for lrt significance is .05. Level of support for Bayes Factor is after Kass and Rafery

(1995).

141