The Evolution of Luminescent Courtship Signals

in Caribbean Ostracods

Honors Thesis

Presented to the College of Agriculture and Life Sciences,

Ecology and Evolutionary Biology Department

of Cornell Univeristy

in Partial Fulfillment of the Requirements for the

Research Honors Program

by

Hilary Suzanne Curran Kates

May 2011

James G. Morin

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Abstract

Males of a unique group of Caribbean ostracods use luminescence in their mating process. These ostracods show high microhabitat specificity and complex extracellular species- specific luminescent courtship displays, which result in a symphony of firework-type displays over shallow Caribbean reef systems after nightfall. Each ostracod’s mating signal consists of a train of light pulses composed of an initial slow “call phase” and a terminal targeting “trill phase” of regular, more rapid and shorter, narrowly spaced pulses. Ostracod displays play a crucial role in sexual selection and therefore speciation in this group. We examined which characteristics of these displays might be used by individuals among three clades of luminescent Caribbean ostracods (Photeros-group, Kornickeria-group and ‘H’-group, based on morphological and molecular work) to differentiate conspecifics from other species. We hypothesized that the more variable initial phase characteristics used in species recognition are more important in shaping the evolution of Caribbean ostracods than those characteristics that make up the constrained trill phase. We combined frame-by-frame analyses of videos of displays from the field for two undescribed species with analyses of 22 species based on published data. We overlaid our findings onto an independently derived phylogeny based on molecular and morphological data of 14 of these 24 species to determine possible signal evolution patterns. Our findings suggest phylogenetic signatures in 5 aspects of the display signal: the duration of the complete signal train, duration and length of the initial phase, the number of pulses in the trill phase, and the amount of time between consecutive pulses in the trill.

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Introduction

Communication whether it is visual, auditory, tactile or olfactory is pervasive in nature and is usually involved in evolution by sexual selection. Furthermore, communication plays integral functions in the everyday lives of most animals, and includes evading predators, luring prey, outcompeting conspecifics, and attracting mates (Bradbury and Vehrencamp 1998). For communicating animals, variation in signals can involve morphological, biochemical or physiological mechanisms; none of these are mutually exclusive (Morin 1986).

The effectiveness of a signal partly depends on the ecological environment in which it is used. For example, tactile communication can only be performed in close proximity. Auditory communication is widespread in aquatic environments where visual signaling may be attenuated by the aqueous medium (Bradbury and Vehrencamp 1998). Visual signaling is generally more prevalent in species that are active in diurnal conditions than dark conditions.

However, signals involving luminescence can be important to organisms that are active in low or no light conditions (Gerrish et al. 2009).

Bioluminescence is often used in inter- or intra-specific communication: whether for confounding a predator, falsely attracting prey, or advertising to potential mates (Bradbury and

Vehrencamp 1998). Numerous studies have examined such signals that evolved as a result of selection pressures, including courtship displays. Luminescent courtship in fireflies (Lewis and

Wang 1991; Branham and Greenfield 1996; Cratsley and Lewis 2003; Lewis et al. 2004) and acoustic courtship signals in insects, amphibians, fishes and birds (Morton 1975; Reinhold 2009;

Sullivan and Kwiatkowski 2007) are well studied systems in which sexual selection, acting on communication signals, has led to species-specific signals. In fireflies, both males and females produce luminescent displays, though it is female choice of male signal characteristics that leads to a mating pair (Lewis and Wang 1991). In firefly displays there are clear temporal and spatial

3 differences among males (Lewis et al. 2004). Females choose mates based on pulse characteristics such as pulse duration and flash rate in order to discriminate among potential mates (Lewis et al. 2004).

Luminescence, has also been identified in a number of intraspecific communication systems across a number of species including fishes, polychaete worms, fireflies and ostracods

(Markert et al. 1961; Morin 1986; Lewis et al. 2004). Male and female polychaete worms signal using luminescence to attract each other in the nighttime marine water column (Markert et al.

1961; Tsuji and Hill 1983). Studies on fireflies have shown that males produce specific trains of light pulses to signal to mates their location and sexual receptivity (Lewis and Wang 1991; Lewis et al. 2004). Similarly, male Caribbean ostracods in the ostracod family Cypridinidae, use luminescent pulses to signal their location to receptive females during courtship (Morin 1986).

Approximately 60% of the roughly 250 species of cypridinid ostracods are bioluminescent. Bioluminescence appears to be a derived trait used as a predator deterrent in all of these species (Morin 1986) (Fig.1). Within this clade, about half of the luminescent species (about 70 species) have been shown to use their luminescence for courtship as well

(Morin 1986; Morin and Cohen 1991; Cohen and Morin 2003; Rivers and Morin 2009) (Fig.1). It is hypothesized that courtship displays evolved from this pre-existing anti-predatory function of the luminescence because more basal species use luminescence only for deterring predators (as far as we know) and all signaling species also use their luminescence to deter predators (Morin

1986, Morin and Cohen 1991).

The mate-signaling luminescent group appears to be restricted to the Caribbean.

Within Caribbean reefs, marine ostracods show extreme microhabitat specificity and in some instances up to 13 species can be found signaling above a given reef system each in its own microhabitat. This kind of spatial specificity implies that species-specific courtship displays are

4 under sexual selective pressure and probably exist for males to be recognized and selected by conspecific females.

Each ostracod display train shows two phases. First, there is an initial “call” phase, of longer duration pulses, which evidently alerts a receptive female to the presence of a conspecifics male (Morin and Cohen 1991). This phase merges into the second trill phase in which pulse interval and duration are shorter and more regular. The trill phase appears to provide the receptive female with a target so that she can intercept the signaling male for copulation (Morin and Cohen 1991).

This signaling group of cyprinidid ostracods contains three major clades, based on morphology and molecular data (Torres and Gonzalez 2007, Gerrish and Torres unpublished): 1- the Kornickeria-group, 2-the Photeros-group, and 3-the ‘H’-group (an undescribed genus) (Fig.

2). Within this group, three broad patterns of courtship displays are known (Morin 1986) (Fig. 1 and 2). The first pattern is a slow vertical shortening display, thought to be the ancestral display, seen in all three clades. This display type has widely spaced pulses that subsequently narrow throughout the display, but become more regular between trill pulses. A second signal pattern, found only in the Photeros-group clade, is the rapid flashing vertical display. This display is defined by individual pulses that persist briefly and typically fade before the next pulse is produced. There is generally less of an abrupt change in pulse characteristics between the initial phase and trill phase in rapid flashing displays. The third pattern is the horizontal display, which is found only in the ‘H’-group clade. This pattern has a reduced initial phase, a large number of similar pulses in the trill phase, and a distinct change between phases. Thus these signal displays are made up of numerous behavioral and ecological characteristics that can be quantified by careful observations and light-intensified videos and then separated into distinct

5 temporal, spatial, train and pulse categories (Morin and Cohen 2010) (Table 1, see also Fig. 3 for visualization).

Based on these broad categorizations, there is clearly considerable variation in ostracod signals among species, but they have not been systematically examined for finer scale patterns that might emerge by overlaying train and pulse characteristics on an independent phylogeny

(Fig. 2). Here I overlay the individual courtship signal characteristics on this phylogeny, and ask whether I can discern any patterns, among clades and within species of those clades, to indicate possible sequences involved in the evolution of the different displays.

Hypotheses: The Photeros-clade consists of both flashing and vertical displaying species, while the ‘H’-group clade consists of both horizontal and vertical displaying species. I therefore hypothesized that the flashing and horizontal displays evolved from a slow vertical displaying ancestor in the Photeros-group and ‘H’- group clades, respectively. I hypothesized that specific train and pulse characteristics may have differential importance to receptive females or competing males. Since the initial phase is proposed to be involved in species recognition

(Morin 1986), I hypothesized that the time and distance characteristics of the initial phase will be more variable among the three clades than those of the trill.

I addressed signal evolution and its overlap on the existent phylogeny on two levels. At the clade level, I provide concrete statistical analyses examining differences among the three clades. At the species level, within clades, I predict the placement of each species examined in this study by comparing the temporal, spatial, train and pulse characteristic data to those characteristics of species with known placement on the phylogeny.

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Methods

Temporal, spatial, train and pulse characteristic information for 24 species of Caribbean luminescent ostracod species were compiled (Table 1, see Fig. 3 for visualization of these characteristics). For 22 of the species these data were extracted from published literature

(Cohen and Morin 1986, 1989, 1993, Morin and Cohen 1988, 2010). These 22 species represent the three clades of ostracods that use luminescence for courtship. The Kornickeria-group species (n=4) are K. coufali, K. hastingsi, K. marleyi and K. louisi; the Photeros group species

(n=8) are P. shulmanae, P. annecohenae, P. morini, P. jamescasei, P. johnbucki, P. mcelroyi, P. gramnicola and Enewton harveyi; and the ‘H’-group species (n=12) are ‘H’ psammobia, ‘H’ ignitula, ‘H’ noropsela, ‘H’ scintilla, ‘H’ micamacula, ‘H’ lucidella, ‘H’ contragula, ‘H’ kuna-up, ‘H’ kuna-down and ‘H’ mizonomma. Two more undescribed species were quantified from the original tapes and included in this study.

Table 1. Quantifiable luminescent ostracod display characteristics used in this study.

Temporal Spatial Train Pulse start of display relative to height above the substratum train pattern duration of each pulse time of sunset total display period substratum type train direction interpulse intervals duration time between display location on the reef train duration interpulse distances repetitions degree of entrainment number of males associated train length relative intensity of each pulse between displays with each display spacing between entrained number of number of initial phase pulses displays pulses per train number of trill phase pulses

Frame-by-frame analyses. Frame-by-frame analyses were conducted on videos taken in

Belize to yield train and pulse characteristic information for the two undescribed species of ostracod: ‘H’ MSH (Massed Shallow Horizontal) and ‘H’ MWU (Mid-Wide Uppers). Exact methods on how these videos were obtained are described in Morin and Cohen (1988). To facilitate clearer resolution of the displays and provide a permanent record of the signals, video recordings were played through a VHS machine and projected onto a white board approximately

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2 meters away and mapped onto paper spanning the projection area. The analyzed signals were pre-selected before quantification as recognizable displays of the target species. Fourteen and

18 individuals were quantified and synthesized into one representative signal for ‘H’ MSH and

‘H’ MWU, respectively.

Each individual signal was quantified in terms of the following information: pulse start times; pulse end times; pulse durations; interpulse intervals, the time between the start of a consecutive pulse and the end time of the previous pulses; interpulse distances, the physical distance between consecutive pulses and the cumulative interpulse intervals and distances (Fig.

3). Interpulse distances were made by marking individual pulses on the white board at one second intervals throughout the display and then averaging them for that particular interpulse number. Each frame (=33 ms) was converted to a time base in seconds.

Frame-by-frame analysis of the tapes gave us only relative spatial distances because the exact distance between the camera and the signal varied among displays. Therefore, relative spatial distances were corrected to absolute distances from actual in situ measurements of the gaps between the trill pulses (which were quite constant) in a train. Individual signal information was then synthesized into one spreadsheet by averaging the pulse durations, interpulse intervals and interpulse distances for each individual pulse across a series of displays.

In some cases, the entire duration of a pulse was impossible to measure (i.e. it had started before the recording or ended after); those pulses were not included in determining the representative signal pattern. Both species were also quantified in terms of temporal and spatial characteristics (Table 1) based on observational data recorded immediately after each field session by James Morin.

Statistical comparisons were made among the three clades based on fifteen display characteristics representative of the train, initial phase and trill portions of an ostracod

8 courtship display. Each species within a clade acted as an individual data point representing that clade. The signal characteristics compared were: train: direction, length (cm), duration (s), number of pulses; initial phase: length (cm), duration (s), number of pulses, duration of first pulse; and trill phase: length (cm), duration (s), maximum number of pulses, mean pulse duration (s), mean interpulse interval (s), mean interpulse distance (cm) and apparent swim speed (cm/s). Apparent rather than actual swim speed was analyzed because ostracods swim in a helical pattern so their actual swim speed is greater than that indicated by their luminescent pulses (Rivers and Morin 2008). Without knowing how large the diameter of this helix is and how tightly each species swims in this pattern we cannot quantify the actual swim speed from video data. Due to unequal sample sizes, a Kruskal-Wallis, nonparametric one-way ANOVA test was run for these comparisons.

To explore finer scale patterns of courtship signaling in the evolution of this group I mapped them on a phylogeny. This well-supported, independently derived phylogeny based on molecular and morphological data was provided by Gerrish and Torres (unpublished, see also

Torres and Gonzalez 2007) (Fig. 2).

Results

Frame-by-frame analysis of two undescribed species from Belize: ‘H’ MSH (Fig. 4): This species signals 1-2 meters up in the water column and over sand channels between coral spurs or patches in Belize (unpublished field observations). Based on analyses of 12 individuals, this species produces narrowly spaced, horizontal displays with an initial phase of 3-4 pulses and a trill phase of between 10 to more than 40 pulses, but usually between 10-30 pulses (Fig. 4D).

The pulse duration (Fig. 4A), interpulse interval (4B) and interpulse distance (Fig. 4C) all decline during the initial 3-4 pulses and then becomes more constant for subsequent trill pulses. The

9 apparent swimming speed for this species was about 3 cm/s. The maximum train length (based on the maximum number of pulses observed) was nearly 42 cm and the maximum train duration was over 17 s. Pulse duration ranged from 3 s (1st) to 1.6 s (mean for trill, n=38); interpulse intervals were fairly short, ranging from about 1 s (1st) to a third of a second (mean for trill, n=38); and interpulse distances were also fairly short, ranging from 1.6 cm (1st) to 1 cm (mean for trill, n=38).

‘H’ MWU (Fig. 5): This species signals 20-100 cm over low coral patches in the shallow backreefs (1-5 m depth) in Belize (unpublished field observations). Based on frame-by-frame analyses of 18 individuals, this species produces 3 - 5 widely spaced initial pulses frequently followed by a trill phase of 5-10 pulses, all in a vertically shortening, upward pattern (Fig. 5D).

The pulse duration (Fig. 5A), interpulse interval (Fig. 5B) and interpulse distance (Fig. 5C) between pulses declines with subsequent pulses, with the most rapid decrease occurring within the first five initial phase pulses, and those thereafter taper off more slowly. The apparent swimming speed of this species is rapid, about 7.8 cm/s. The maximum train length (based on the maximum number of pulses observed) was over one meter with a display duration of about

18 s. Pulse duration was initially quite long, > 4 s, and the trill duration was about less than half of this duration, <2 s (n=10). Interpulse intervals ranged from about 2.7 s (1st) to about 0.8 s

(mean for trill, n=10); and interpulse distances were wide and ranged from >12 cm (1st) to <6 cm

(mean for trill, n=10) (Appendix 1-3).

Tools for comparison. A spreadsheet was generated of the various temporal, spatial, train and pulse characteristics of each species (details in Appendix 1-3). Pulse characteristics of each species were individually plotted: pulse duration, interpulse interval and interpulse distance (Appendix 4). These data formed the basis for compiling the time-distance figures for each species (Appendix 4, Fig. 4 and 5). These time-distance figures were created by plotting

10 the cumulative interpulse interval data plus the pulse duration by the cumulative interpulse distances for each pulse (Appendix 4, Figs. 4 and 5). A similar plot was generated per clade by flattening all of the species time-distance plots into one figure (Fig. 6, 7,8A and 8B). Likewise, a spatial display was generated based on height above the substrate at signal start, the direction of the signal and the interpulse distance information for each species (Fig. 9). The horizontal spacing location shown in Fig. 9 is arbitrary, except for the horizontal/obliquely displaying species, where horizontal and vertical distances are spatially relevant. These figures and tables facilitated both statistical clade comparsions as well as within clade, species comparisons.

Overall clade characteristics. These results are based on the range of values seen in each measurement based on the species that are found in that clade. Characteristics are broken up into overall train, initial and trill phase characteristics and do not represent individual pulses unless specified.

The Kornickeria-group clade (Fig. 6, 9A) (n=4). The basal clade, i.e. the genus

Kornickeria, includes only species with vertical shortening displays, but they occur in either an upward (n=2) or downward (n=2) direction (Fig. 6). In terms of height above the substratum, the species’ displays overlap, however initial pulses of a given species do not overlap (Fig 9A).

All four Kornickeria species consistently produce trills. The apparent swim speed of this clade ranges from 2-7 cm/s. The total pulse number ranges from 18-25; train length, 15-45 cm; and train duration, 6-19 s. The initial phase characteristics range from 3-5 in pulse number; <3-<12 cm in length; and about 3->8 s in initial duration, and for trill characteristics: 15-20 in pulse number; 12-35 cm in length and 4-14 s in trill duration. The duration of the 1st pulse ranges from 2-6 s, while mean trill pulse duration ranges from <1-3 s. Mean interpulse intervals range from 1/5-3/4 s and mean interpulse distances range from ¾- 2 ¼ cm (See Appendix 1-3 for specific values for Kornickeria-clade species).

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The Photeros-group clade (Fig. 7, 9B) (n=8). The Photeros clade includes species with both slow vertical shortening displays in either the upward (n=2) or downward (n=2) direction and rapid flashing displays in upward (n=2) or downward directions (n=2). All eight Photeros clade species consistently produce trills, but the initial phase varies among species (for flashing species the change between initial phase and trill phase is not always distinct). The apparent swim speed of this clade ranges from 3-16 cm/s for flashers to 0.8-6 cm/s for vertical shortening displayers. The total number of pulses in the train is generally fairly low ranging from 8-20; train length: 20 cm – 1 ½ m; and train duration: 5->25 s. The initial phase characteristics are: number of pulses: 3-6; length: 0 cm-1 ¼ m; duration: 1-24 s. The trill phase characteristics are: 4-15 pulses; length: 9 cm-<1 ½ m; and duration: 2-< 25 s. The duration of the 1st pulse ranges from

1/8 -7 s, while mean trill pulse duration ranges from 0.2 – <25 s. Mean interpulse intervals range from 0.5 ->1 ¼ s and mean interpulse distances range from 0.6-2 cm (See Appendix 1-3 for specific values for Photeros-group clade species).

The ‘H’-group clade (Fig. 8, 9C) (n=12). The ‘H’-group clade shows enormous variation in display patterns including species with slow vertical shortening displays in either the upward

(n=4) or downward (n=4) directions, horizontal displaying species (n=3), and one species, ‘H’ psammobia, that first displays horizontally and then vertically upward (Fig 4A, 4B and 7C).

Seven out of the twelve ‘H’-group species consistently produce trills, while the other five do not.

The apparent swim speed of this clade ranges from 1.0-8 cm/s. The ‘H’-group clade also varies widely among species in terms of overall train characteristics: total number of pulses: 9-256 (or more); length: 6 cm-2.5 m (or more); and duration: 12 s- >1 min. The initial phase characteristics are as follows: number of pulses: 1-6; length: 0-<45 cm; duration: 7->16 s. The trill characteristics are: number of pulses: 6-250 (or more); length: >4-225 cm; and duration: 6 s-

>1min. The duration of the 1st pulse ranges from 2-8 s, while mean trill pulse duration ranges

12 from 1-<5 s. Mean interpulse intervals range from 1-<5 s and mean interpulse distances range from 0.5 -<5 cm (See Appendix 1-3 for specific values for ‘H’ group clade species).

Statistical comparison of signal characters among clades. There were no statistical differences among the clades for 9 of the 15 analyzed characteristics. No differences were found in train length (cm), total number of pulses per train, mean pulses in initial phase, duration of first pulse, mean pulse duration in trill (s), mean interpulse distance in trill (cm), total trill duration (s), total trill length (cm) and apparent swim speed (cm/s).

However, a few characteristics did show a trend (i.e. a p-value between 0.05-0.1). First, the mean trill interpulse interval (p= 0.098, df=2), for the Kornickeria-clade was shorter than for all species combined. Second, the maximum number of pulses per trill (p= 0.053, df=2) for the

Kornickeria-clade and Photeros-clade had fewer pulses than for all species combined. Third, the train duration (s) (p= 0.098, df=2) of the Kornickeria-clade was shorter than for all species combined. Fourth, the initial phase length (cm) (p= 0.062 df=2) of the Kornickeria-clade was shorter than for all species combined. Finally, the initial phase duration (s) (p= 0.099, df=2) of the Kornickeria-clade was shorter than for all the species combined.

Discussion

Over 60 new species in at least four new genera of luminescent cypridinid ostracods have been (n=24) or are being described (n=36) from the Caribbean Sea. In all of these species, males produce species-specific luminescent courtship displays. Several studies on luminescent ostracods have argued that the luminescent courtship signals produced by these organisms are under strong sexual selection, which has resulted in the rapid evolution of these species within this group. As a result there is a high diversity of luminescent species found only in the

Caribbean, each with its own narrow habitat range and specific signal display (Morin 1986,

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Morin and Cohen 1991, 2010, Cohen and Morin 1993, Rivers and Morin 2008). At any given location up to 13 species can be found signaling in a given ecosystem, each with its own spatio- temporal microhabitat, yet each species signal appears to be strongly conserved (Morin and

Cohen 2010); such specific signal fidelity provides evidence that some selective force must exist to allow these organisms to live in sympatry within the reef ecosystem and still recognize individuals of their own species. While species within and across clades show narrow limits, collectively they occupy a broad range of spatial and temporal niches (Fig. 9). However, there are a few cases where the displays of two species do occupy most of, if not entirely, the same spatial and temporal niche. Especially in cases such as these, discrete differences between train pulse patterns may be used to identify individuals of the same species.

If this is so, then pattern recognition by con- and heterospecifics is important in driving the evolution of a particular species’ signal. But what particular patterns are under this selection? Our goal was to identify potential characters by carrying out statistical analyses of characteristics across the three clades, but also more specifically by a comparative analysis of the characteristics across a variety of species. It is important to note that this was our initial attempt to identify characters, and though there are an exhaustive set of characters that could be examined, time constraints limited us. Nonetheless, the results we found have brought to light several interesting trends that warrant further analyses.

Specific clade level differences. None of the train or pulse characteristics analyzed showed statistically significant differences among clades. Sample sizes (24) are fairly small so the addition of more of the 36 undescribed species could provide greater insights. First,

Kornickeria, the basal clade, demonstrates more constraint in several key characters than the other two clades. Generally the train and pulse characteristic means of the Kornickeria-group were lower than for all species combined. These characters included the mean trill interpulse

14 interval, maximum number of pulses per trill, total train duration, initial phase length and duration. Interestingly, these trends encompass all four parts of a typical ostracod signal: train, initial phase, trill phase and individual pulse characteristics. So while we cannot conclude that any one of these four signal parts is more or less important in the sexual selection pressure that leads to speciation in this group, the pattern is interesting and warrants further analysis.

Another interesting trend was a higher number of pulses seen in the trill phase of the

‘H’-group compared to all species combined. The higher number in the ‘H’ group species is probably affected by the species with horizontal displays, which tend to produce very high numbers of trill pulses (Fig. 8C). Furthermore, Kornickeria species are typically quite shallow water species overall so they might have less space to display compared to horizontal displaying species, which typically display along extensive sand channels. Thus, horizontal displaying species have virtually extensive, unlimited space to display, while some vertical displayers are limited by the depth of the water.

Shallow water depth for displaying could explain why the total train duration of the

Kornickeria-clade is also lower than the mean, but would beg the question why there is not also a trend in the Photeros-clade for this same parameter. However because Kornickeria species have lower values in these characteristics, the ancestral vertical displays might have had characteristically shorter train lengths and durations, while derived forms have longer, lengthier displays. It will be interesting to examine this further and compare only the vertical displaying species in each of the clades for these same characteristics (time did not permit me to do so).

Not surprisingly, the initial phase duration is shorter in the basal clade, Kornickeria, whereas the trill phase duration does not vary among the clades. As I hypothesized the initial phase is likely more variable because the animals are using it as a cue for species-specific information, so it should not be as constrained as the trill, which is used for targeting and

15 intercepting conspecifics once species recognition has been determined. The length of the initial phase in the Kornickeria-clade was also shorter than the mean across all of the species evaluated. This result provides more evidence that the initial phase, being the most divergent part of the display, has a significant influence on the evolution of ostracods.

Based on these findings, several conclusions can be made about the distinctions among the three clades in terms of courtship displays. The Kornickeria-clade is not only constrained to vertical signals, but it is also constrained to lower values compared to the other clades in the timing between subsequent pulses of the trill phase, maximum number of pulses per trill, total train duration and initial phase duration and physical length of the initial phase.

The molecular phylogeny and species overlay predictions. Based on my findings and the prior molecular and morphological studies, I am able to discern patterns in the evolution of

Caribbean luminescent ostracods based on courtship displays (Fig. 2). It is important to note on the phylogeny, based on combined molecular and morphological characters, that while initial branches are quite well resolved, some of the terminal ones are not. Further, the cladogram includes only 14 of more than 60 known species and does not include 10 of the species included in this study, so only tentative conclusions can be drawn.

So what can we infer about signal evolution based on the partial cladogram (Fig. 2) of 14 of the 24 species presented in this study? Further, what can we predict about where the species might be inserted based solely on our display data? Theoretically speciation (genetic divergence) can occur either sympatrically or parapatrically (Cain et al. 2008). The analysis of these ostracod signals in conjunction with a robust phylogeny can shed light on which of these alternatives (neither of which is mutually exclusive of the other) might be operating in this system. We know that the signaling location and time for a given species is very precise, so presumably species are very constrained, but we do not know how much flexibility there is in

16 these various parameters (i.e. characteristics in Table 1). Is habitat for signaling more or less flexible than direction of signal, or number of pulses, or pulse duration? A more thorough phylogeny of all species that is based on molecules and morphology can be used to test these alternatives.

Sister taxa of vertical displays. One striking pattern among the vertical shortening display species is a fairly even split between upwardly and downwardly produced displays in all the clades. There appears to be no distinct pattern within the clades (Fig. 2 arrows). However upon closer inspection, sister taxa generally show opposing train directions (see also other species below). This signaling direction could be one plausible mechanism of divergence and consequently speciation in sister ostracod species.

Currently, we detect a range of differences for both possible sympatric and allopatric speciation in this system. For instance, there are few if any morphological differences between

‘H’-group species kuna-up and kuna-down from Panama and Cohen and Morin (1988) have accordingly placed them in the same species, though there are no comparable molecular data.

They are presumably sibling species. Both signal above corals, but with some microhabitat differences (kuna-up over shallow reefs near drop-offs, kuna down over deeper coral slopes but adjacent to one another and sometimes overlapping i.e. they are sympatric Appendix 1) and they have significant (opposing) signal patterns (kuna-up with long, upward and kuna-down with short, downward).

On the other hand, while most apparent sister taxa show similar or opposite directions of display, but occur in distinct geographical locations, which would support allopatric speciation scenarios. For instance, K. hastingsi has been described as two subspecies by Cohen and Morin

(1993) with K. hastingsi hastingsi from Jamaica and K. hastingsi carriebowae from Belize. They are similar morphologically and in their displays (habitat, patterns, timing). Contrastingly, other

17 pairs from different locations with similar habitat patterns show distinct display differences. For example, P. gramnicola from Panama is a grassbed species as is P. annecohenae from Belize.

Their displays are similar (both produce upward flashing displays above grassbeds, but the details of their displays are different. Another example is P. mcelroyi from Jamaica and P. morini from Belize. Both are reef flashers in the same habitat, location and time, but P.mcelroyi is an upward displayer and P. morini is a downward displayer. Clearly both habitat and location are plausible mechanisms for these hypothesized sister taxa pairs, but the true driver of these relationships is yet unclear. If spatial proximity is the driver among these Photeros species then sister taxa are P. morini and P. annecohenae, but if habitat is the driver then sisters are P. gramnicola and P. annecohenae. Other potential sister pairs include ‘H’ ignitula and ‘H’ psammobia; P. shulmanae, P. jamescasei and P. johnbucki; K. louisi and K. coufali; ‘H’ micamacula and ‘H’ lucidella; and ‘H’ MSH and ‘H’ noropsela. However, it is beyond the scope of this thesis to analyze these differences more deeply, but it does point the way to fruitful research in the future.

Vertical display species predictions. Based on the current phylogeny, vertical shortening displays are ancestral to both horizontal and flashing displays. If flashing has derived once, then a resolution of the polychotomy of the four Photeros species listed (Fig. 2) would then place P. shulmanae as basal to the three flashing species (dotted line in Fig. 2) and the flashing species would form a monophyletic group.

Flashing display species predictions. Based on my observation that sister taxa tend to display in opposite directions and assuming that sister taxa evolve via sympatric divergence, I would conclude that P. morini is likely more closely related to P. annecohenae than to P. gramnicola. Both P. morini and P. annecohenae are found in Belize, while P. gramnicola is found in Panama.

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Horizontal displays. Solely based on our findings of the courtship displays, horizontal displays appear to be a derived state that represents a unique switch in a number of signal parameters, especially trill length (basically these displays are highly trill dominated). Thus we initially hypothesized that these species would be a monophyletic group nested within the ‘H’ group clade. However, the molecular phylogeny indicates that they are not monophyletic.

Horizontal displays appear to have independently arisen at least twice in the ‘H’-group clade.

Based on the cladogram, there also would be a reversion back to the vertical pattern in ‘H’MWU and its sister taxon ‘H’ scintilla. If horizontal displays represent a major switch as we suspect, then a more parsimonious explanation should predict that ‘H’ noropsela will ultimately prove to be sister to ‘H’ MSH (dotted line in Fig. 2). A more detailed analysis of this clade with more markers will resolve this issue. Resolving this issue will indicate much about how plastic display patterns are in ostracods. Further analysis of train and pulse characteristics in relation to other display characteristics such as temporal and spatial patterns may reveal finer resolution about how some of these polychotomies will be resolved.

Predictions on the placement for the other 10 species in the phylogeny.

Kornickeria clade: Kornickeria coufali and K. louisi clearly belong in the Kornickeria- group based on major morphological similarities. All occur in Jamaica and show distinct habitat and display patterns. Currently, based on the discussion above, we cannot predict relationships among these four species.

Photeros clade: If, as we predict, the resolution of the polychotomy of Fig. 2 shows that flashing displays form a monophyletic group within the bigger clade, then E. harveyi, P. jamescasei and P. johnbucki will be basal in the clade along with P. shulmanae. E. harveyi has been placed in a separate genus by Cohen and Morin (2010) and has been placed at the base of

19 this clade. We cannot predict the relationship of the other vertical shortening species at this time.

‘H’-Group Clade: Horizontals. As with the Photeros clade situation, accurate placement of ‘H’ group species must wait until more molecular and morphological data are accumulated to resolve whether horizontal displays are monophyletic or not (see discussion above).

‘H’noropsela, a horizontally signaling species (Fig. 8C) is closely related to the horizontally signaling ‘H’ MSH species based on courtship displays. Based on the molecular phylogeny, ‘H’ noropsela is more related to the reverted vertical species, ‘H’ MWU and ‘H’ scintilla. One hypothesis that can be drawn from these opposing results is that subsequent molecular and morphological work may reveal that ‘H’ noropsela has actually diverged from ‘H’ MSH.

Therefore, no reversion back to the ancestral state would need to be made for ‘H’ MWU and ‘H’ scintilla and thus an even more parsimonious tree would be formulated by also accounting for the role courtship displays have on sexual selection in these ostracods.

Verticals. It is currently unclear where the remaining species, ‘H’ psammobia, ‘H’ lucidella and ‘H’ ignitula will place in the ‘H’-group. Morphologically, ‘H’ kuna-up and ‘H’ kuna- down are virtually identical, so we would predict that they are sister taxa and that they are sister to ‘H’ mizonomma. ‘H’ lucidella and ‘H’ ignitula have patterns comparable to those at the base of the ‘H’ group and will likely insert among them. ‘H’ psammobia, however, has quite a complex display pattern so may insert somewhere among the terminal taxa. It could be an interesting case study for the switch between vertical displays and horizontal since it has elements of both.

Current analyses were skewed due to the large variation brought upon by horizontal and flashing displays. The next step should be to reevaluate only the slow vertical shortening displays across all clades. And although 24 species were examined, the examination of more

20 species within each clade should produce clearer trends and a more comprehensive understanding of how the courtship displays truly impact the evolution of this group of organisms.

Conclusions

With my in-depth analysis we now know that some display characteristics are clearly conserved across the clades while others are more variable. We can now focus on those characteristics that vary to determine how courtship displays have shaped the evolution of these species; but the patterns are complex.

An analysis of courtship display characters on 24 signaling ostracod species from the

Caribbean has revealed:

General patterns:

Courtship displays show a broad range of variability among, but not within species

(based on my own video observations and unpublished field observations), and can

occur in upward, downward, long or short, horizontal or flashing patterns.

Morphological and molecular data indicate three monophyletic clades (Torres and

Gerrish unpublished): Kornickeria (n=4), Photeros (n=8) and ‘H’ group (n=12) in this

study.

All three clades have species with vertical shortening displays. This display pattern

appears to be the ancestral display type.

Morphological and molecular data (Torres and Gerrish unpublished) place the

Kornickeria-clade as basal to the Photeros and ‘H’-group clades; these species only

produce vertical shortening displays.

21

The Photeros-clade contains both vertical shortening and flashing display species, but

flashing displays are unique to the Photeros-clade; we predict that flashers form a

monophyletic group within the larger Photeros-clade.

The ‘H’-group clade contains both vertical and horizontal displayers and horizontal

displays are unique to the ‘H’-group. However, based on morphological and molecular

data the horizontal displayers do not appear to form a monophyletic group.

Specific patterns:

The basal Kornickeria-clade shows a trend toward:

-shorter total train duration

-shorter initial phase length

-shorter initial phase duration

-fewer number of pulses per trill

-lower mean trill interpulse interval

The Photeros-clade shows a trend toward a fewer number of pulses per trill.

Future directions:

The displays of more species need to be examined to determine the validity of these

trends.

Trends based on the temporal and spatial characteristics suggest sister taxa may diverge

differentially between the clades via both sympatric and allopatric mechanisms.

More molecular and morphological information is required to resolve the evolution of

signal patterns in these ostracods.

22

These data indicate the finer scale trends that have occurred within this signaling clade of cypridinid ostracods. This comprehensive compilation of patterns indicates where some changes have occurred and which characters have been constrained. By determining which characteristics are constrained and which are variable, we have begun to understand which traits are undergoing selection and therefore probably directly involved in speciation of these ostracods. Understanding the variation in the ancestral display will elucidate the characteristics that define the other display types, horizontal and flashing, and by what mechanisms they diverged from the ancestral signal form. This comparative approach gives us a more comprehensive understanding of the role of luminescent courtship in the evolution of Caribbean ostracods.

23

Acknowledgments

My biggest acknowledgment is to Jim Morin, my advisor and mentor, for taking the time to teach me about the fascinating displays of Caribbean ostracods, fostering my desire to do research. Likewise, he has fostered and supported me full-heartedly as a young ecologist since the day I first asked him to be my advisor, which is now a long time ago! Thank you immensely for the time and effort you put in to teaching me and working with me. Thank you also to Myra

Shulman and Laura Eireman for their support and statistical guidance with this project. Thank you to Cornell University and all of the staff members of the honors program, particularly to

Laurel Southard, Colleen Kearns and Kelly Zamudio for their encouragement throughout the entire process. And finally a warm thank you to my family and friends who have listened to me rattle on about ostracod courtship signals and supported me through thick and thin.

24

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Gerrish, G.A., J.G. Morin, T.J. Rivers and Z. Patrawala. 2009. Darkness as an ecological resource: the role of light in partitioning the nocturnal niche. Oecologia 160: 525-536.

Gerrish, G.A. and E. Torres. 2010. A systematic revision of bioluminescent Caribbean ostracods (Cypridinidae) and the role of sexually selected courtship displays in speciation. (unpublished NSF grant proposal)

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Morin J.G., and A. C. Cohen. 2010. It’s All About Sex: Bioluminescent Courtship Displays, Morphological Variation and Sexual Selection in Two New Genera of Caribbean Ostracodes. Journal of Crustacean Biology 30(1): 56-67.

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Sullivan, B.K. and M.A. Kwiatkowski. 2007. Courtship displays in anurans and lizards: theoretical and empirical contributions to our understanding of costs and selection on males due to female choice. Functional Ecology 21: 666-675.

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Torres, E., and J.G. Morin. 2007. Vargula Annecohenae, a New Species of Bioluminescent Ostracode (Myodocopida: Cypridinidae) from Belize. Journal of Crustacean Biology 27(4): 649-659.

Torres, E., and V.L. Gonzalez. 2007. Molecular Phylogeny of Cypridinid Ostracodes and the Evolution of Bioluminescence. Proceedings of the 14th International Symposium on Bioluminescence and Chemiluminescence: Chemistry, Biology and Applications World Scientific Publishing Company, New Jersey: 269-272.

Tsuji, F., and E. Hill. 1983. Repetitive Cycles of Bioluminescence and Spawning in the Polychaete, Odontosyllis phosphorea. Biol. Bull. 165(2): 444-449.

26

Fig. 1 General phylogeny of the evolution of ostracod luminescence. There are 250+ species of ostracods in the family Cypridinidae; 60% of those have luminescent capabilities used for anti- predatory functions (teal and pink branches). Half of these species also use their luminescence for courtship signaling (only pink branches) and are restricted to Caribbean waters. Dotted areas indicate more than one branch and plus signs indicate more species fall into these branchs than are indicated on this simplified phylogeny. (modified from Cohen and Morin 2003)

Fig. 2 Cladogram of the three groups of ostracod species that use luminescence for courtship (Kornickeria-group, Photeros-group and ‘H”-group). Based on molecular 12S + 16S rRNA data and morphological work of Gerrish and Torres 2010 (unpublished and Torres & Gonzalez 2007). Arrows indicate signaling direction. Dotted lines indicate predictions based on courtship displays, not elucidated by the molecular and morphological work (see text).

Fig.3 Comparison of a Caribbean ostracod signal viewed in the field to the time-distance graphic generated by frame-by-frame quantifications. Field view shows three pulses for ‘H’ MWU species which corresponds to the bottom three horizontal lines (as well as numerous ‘H’ MSH displays). On the graphical representation, the furthest dot to the left of each horizontal blue line represents the start time of a pulse; the dot furthest right is the end time of that pulse. The vertical axis on the graphical representation corresponds to the interpulse distances as viewed in the field. The time-distance graph shows the typical number of pulses for a complete signal of that species. In this species only about three pulses are visible at a given time (indicated by the filled dots on the far left of the time-distance graphic, empty dots are pulses to come). The complete signal is called the train; all pulses in a train are not necessarily visible at one time in the field. Train pattern is based on the orientation of the signal in the water column ie. vertically upward for ‘H’ MWU. The reef background in the field view image on the right depicts some of the spatial characteristics ie. signaling height in the water column. (For a more comprehensive understanding of display characteristics see Morin and Cohen 2010.) Image: Jim Morin 4 1.4 3.5 'H' MSH pulse duration 1.2 'H' MSH interpulse interval 3 1 2.5 0.8 2 0.6 1.5

pulse duration (sec) duration pulse 0.4 1

interpulse interval (sec) interval interpulse 0.2 0.5 0 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 A 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 B pulse number interpulse number 2 45 1.8 'H' MSH interpulse distance 40 'H' MSH time-distance 1.6 35 y = 2.856x - 2.3037 1.4 30 R² = 0.9967 1.2 (cm) 1 25 0.8 20

0.6 distance 15

0.4 10 interpulse distance (cm) distance interpulse 0.2 5 0 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 D 0 5 10 15 20 C interpulse number time (sec)

Fig. 4 Graphical view of pulse characteristic measurements for the entire ‘H’ MSH courtship signal (n=12). A-pulse duration versus pulse number; B- interpulse interval versus interpulse number; C- interpulse distance versus interpulse number; D- time-distance relationship (based on cumulative interpulse intervals and distances from B and D). 5 3.5 'H' MWU pulse duration 4.5 3 'H' MWU interpulse interval 4 3.5 2.5 3 2 2.5 2 1.5 1.5 1 pulse duration (sec)duration pulse 1 0.5 (sec) interval interpulse 0.5 0 0 A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pulse number interpulse number

16 120 'H' MWU interpulse distance 'H' MWU time-distance 14 100 12 y = 6.543x - 5.8183 80 R² = 0.9915

10 (cm) 8 60 6 40 4 distance 20 interpulse distance (cm) distance interpulse 2 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 5 10 15 20 C interpulse number D time (sec) Fig. 5 Graphical view of pulse characteristic measurements for the entire ‘H’ MWU courtship signal (n=18). A-pulse duration versus pulse number; B- interpulse interval versus interpulse number; C- interpulse distance versus interpulse number; D- time-distance relationship (based on cumulative interpulse intervals and distances from B and D). Fig. 6 Time-distance relationship of luminescent courtship display trains for four species of the Kornickeria clade. Each pair of similar colored horizontal points represents the start and end time of a pulse. Vertical and horizontal distances between successive points represent the distance the species has moved and over what amount of time, respectively. The slope of the lines represents the apparent swimming speed of the ostracod (but not actual-see text). Arrows represent the direction that each species displays. (generated from data in Cohen and Morin 1993)

Fig. 7 Time-distance relationships of luminescent courtship display trains for Enewton harveyi and seven described species of Photeros. Successive data points for each species represent distance traversed (cm) and time (s) elapsed since the prior pulse. Each pair of horizontal points represents the start and end of a pulse and the horizontal line between (shown only for vertical shortening displays) represents the duration of a pulse. The slope of the line connecting the start of each pulse indicates the apparent (but not the actual-see text) swimming speed. Each open arrow indicates the direction that the display is produced in the field; red arrows indicate upward displays, blue arrows downward displays. Pulse duration lines are offset from zero at the first pulse for clarity of comparison. (from Morin and Cohen 2010)

Fig. 8A Time-distance relationship of luminescent courtship display trains for five species of ‘H’ group clade. Each pair of similar colored horizontal points represents the start and end time of a pulse. Vertical and horizontal distances between successive points represents the distance the species has moved and in what amount of time, respectively. The slope of the lines represents the apparent swimming speed (cm/s) of the ostracod. Arrows represent the direction that species display; black represents trains that continue beyond several hundred pulses and are not visually captured here. (based on new analysis and data from Cohen and Morin 1986, 1988 and 1989)

Fig. 8B Time-distance relationship of luminescent courtship display trains for seven species of ‘H’ group clade. Each pair of similar colored horizontal points represents the start and end time of a pulse. Vertical and horizontal distances between successive points represents the distance the species has moved and in what amount of time, respectively. The slope of the lines represents the apparent swimming speed (cm/s) of the ostracod. Arrows represent the direction that species display. (based on new analysis and data from Cohen and Morin 1986, 1988 and 1989)

Fig. 9. Spatial relationship of three courtship signaling ostracod clades. Bottom represents the substrate over which the ostracods signal. Hash marks on the vertical axis indicate a jump or change of interval on the scale. A-Kornickeria clade species (n=4), B- Photeros clade species (n=8) and C- ‘H’ group clade species (n=12). (generated from new data and data in Cohen and Morin 1986, 1989, 1993, Morin and Cohen 1988, 2010). Appendix 1 General Characteristics of Luminescent Displaying Caribbean Ostracods Apparent Type of Direction Display swimming Clade Species Source* Locality Lat/Long analysis of display pattern speed (cm/s) 18.47N, 77,42W; 18.47N, Kornickeria C+M 1993 Jamaica 77.32W; 18.43N, 77.20W; video analysis down vertical down 3.67 louisi 18.17N, 76.38W 18.47 N, 77.42 W; 18.7N, zig zag C+M 1993 video analysis Group K. coufali Jamaica up 2.16 76.38W; 18.10N, 76.33W upward

short, slightly K. marleyi C+M 1993 Jamaica 18.47 N, 77.42 W video analysis down zigzag 3.11 downward

Kornickeria Belize & 16.80N, 88.08W; 18N, 88W; hastingsi C+M 1993 video analysis up upward 6.96 Jamaica 16.4N, 87.8W

direct Photeros C+M 1986, observations slow, wide, Panama 9.550N, 78.947W down 5.56 shulmanae M+C 2010 and video downward analysis

P. 16.801-16.897N, 8.066- rapid flashing, M+C 2010 Belize video analysis up 5 annecohenae 88.103W up

16.782-16.816N, 87.866- rapid flashing, P. morini M+C 2010 Belize video analysis down 7.27 88.084W down

18.108N, 76.316W, short, slow, Group E. harveyi M+ C 2010 Jamaica video analysis down 0.79 18.172N, 76.381W down

18.472N, 77.410W; moderate P. jamescasei M+ C 2010 Jamaica video analysis up 1.6

18.472N, 77.405 W upward Photeros P.johnbucki M+C 2010 Jamaica 18.472N, 77.405W video analysis down downward arc 5.5

rapid flashing, 18.472N, 77.410W; 18.472 P. mcelroyi M+C 2010 Jamaica video analysis up stationary to 2.67 N, 77.405W up

direct P. observations rapid flashing M+C 2010 Panama 9.557N, 78.953W up 16 gramnicola and video up analysis apparent Type of swimming Clade Species Source Locality Lat/Long analysis direction pattern speed

MSH Belize video analysis horizontal horizontal 2.95

vertical, MWU Belize video analysis up 7.75 widely spaced

L shaped, psammobia C+M 1989 Panama 9.33.14N, 78.55.23W frame-by-frame L-shape horizontal first 1.27 then upward

vertical ignitula C+M 1989 Panama 9.33 14N, 78 55 23 W frame-by-frame down 2 downward horizontal or noropsela C+M 1989 Panama 9 33 14N, 78 55 23 W frame-by-frame horizontal slightly 3.45 oblique

medium scintilla C+M 1989 Panama 9 33 14N, 78 55 23 W frame-by-frame down length vertical 1.11 downward

short micamacula C+M 1989 Panama 9 33 14N, 78 55 23 W frame-by-frame down 1

"H" Group downward vertical lucidella C+M 1989 Panama 9 33 14N, 78 55 23W frame-by-frame up 3.59 upward direct observa- oblique/ oblique contragula C+M 1986 Panama 9 33 14N, 78 55 23W tions and video hori- 2.25 upward analysis zontal direct observa- vertical wide kuna up M+C 1988 Panama 9 33 14N, 78 55 23 W tions and video up 7.8 upward analysis direct observa- vertical, short kuna down M+C 1988 Panama 9 33 14N, 78 55 23 W tions and video down 1.33 downward analysis

direct observa- zigzag oblique mizonomma M+C 1988 Panama 9 33 14N, 78 55 23 W tions and video up 4 upward analysis

*: C = Cohen; M = Morin Appendix 2 Train Characteristics of Luminescent Displaying Caribbean Ostracods Length Duration Number of Pulses

Display

Clade Species Direction (cm)

pattern (sec)

Pulses

Trill (cm) Trill

Trill (sec) Trill

Trill pulses pulses Trill

Train (cm) Train

Train (sec) Train

[maximum]

Total Pulses Total

Initial Phase Initial Phase Initial Phase Initial Kornickeria short, down 14.67 2.89 11.55 5.81 2.78 11.55 18 3 15 louisi downward

Group K. coufali up zig zag 36.19 10.44 24.3 18.93 8.38 37.65 20 5 15

short, slight K. marleyi down 43.16 8.95 34.05 17.72 6.89 44.4 19 4 15 zig zag

hastingsi up fast upward 44.59 11.56 32 8.05 3.56 20.4 25 5 20 Kornickeria Photeros slow, wide, down 135 110 20 24.1 23.7 16 8 4 4 shulmanae downward P. up rapid flashing 35.25 2.5 35 9.67 2.45 2.15 13 3 10 annecohenae

P. morini down rapid flashing 46.5 15 52 5.03 1.23 10.855 16 3 13 Enewton down short, slow 46.5 15 7.5 5.03 1.23 9.5 10 5 5 Group harveyi moderate P. jamescasei up 137 125 22 25 21.75 27.5 17 6 11 upward

P.johnbucki down downward arc 91 36 82.5 25.25 18.5 48.75 20 5 15 Photeros

stationary P. mcelroyi rapid flashing 20 0 30 19.75 13 3.75 20 5 15 to up

P. gramnicola up rapid flashing 146 6 180 14.28 4.9 2.52 12 3 9

horizontal and MSH horizontal 41.82 4.35 38.76 17.38 4.07 61.94 42 4 38+ long

MWU up widely spaced 106.02 44.4 61.6 18.02 11.05 20.79 16 5 11

horizontal to psammobia L-shape 49.3 30.5 36.4 25.65 12.98 32.72 11 3 8 upward vertical ignitula down 36.2 25.2 10 13.22 8.64 15 14 4 10 downward oblique and noropsela horizontal 226.01 26.01 200 68.93 10.7 228 106 6 100+ long medium scintilla down 30.55 15.55 18.75 30.55 15.73 15 20 5 15 length very short micamacula down 6 2.5 3.5 12.3 7 7 9 2 7 downward "H" Group vertical lucidella up 38.1 24.6 13.5 23.02 8.9 15 20 5 15 upward oblique/ oblique up contragula 262.5 84 225 37.3 10 475 256 6 250+ horizontal and long

kuna up up long, wide 173.5 37.5 136 34 10.3 25.5 20 3 17

short kuna down down 26 14 12 18.5 10 6 10 4 6 downward zigzag oblique mizonomma up 128.5 0 116 36 4.5 130.5 30 1 29 upward Appendix 3 Pulse Characteristics of Luminescent Displaying Caribbean Ostracods Pulse 1 Pulse 2 Pulse 3 Pulse 4 Pulse >5 Trill

Clade Species

Pulse#

Trill number Trill

Direction

Pulseduration Pulseduration Pulseduration Pulseduration Pulseduration Pulseduration

Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse

Interpulse Distance InterpulseDistance InterpulseDistance InterpulseDistance InterpulseDistance InterpulseDistance InterpulseDistance Display pattern Displaypattern Kornickeria short, down 2.23 1.39 1.01 (n=5-15) 0.77 louisi downward 1.21 1.52 0.56 1.37 0.33 1 0.21 0.77 zig zag Group K. coufali up 4.44 1.05 1.98 4.21 0.97 2.48 3.56 1.68 2.97 3.65 1.54 3.01 5 3.21 1.47 3.07 (n=10-15) 2.51 0.75 1.62 short, slight K. marleyi down zig zag 5.45 4.54 3.94 3.15 (n=8-15) 2.96 1.86 3.53 0.92 2.69 0.96 2.73 0.8 2.43 0.73 2.27 fast upward hastingsi up 2.07 1.82 1.62 1.47 5 1.13 (n=10-20) 1.02

Kornickeria 0.9 2.8 0.66 3.38 0.52 3.05 0.35 2.33 0.23 2.63 0.23 1.6 Photeros slow, wide, down 7.2 7.2 7.2 7.2 (n=1-4) 4 shulmanae downward 5.5 40 5.5 40 5.5 30 5.5 10 0.9 5 P. rapid up 0.35 0.35 0.35 (n=6-10) 0.215 annecohena flashing 1.05 1.25 1.05 1.25 1.05 1.25 0.7 3.5 rapid P. morini down 0.125 0.125 0.125 (n=7-17) 0.084 flashing 0.55 7.5 0.55 7.5 0.55 7.5 0.55 4 Enewton short, slow down 7 7 7 7 (n=4-6) 1.9

Group harveyi 2.3 5 2.3 5 2.3 5 2.3 5 1.9 1.5 P. moderate up 8 8 8 8 5 8 (n=5-14) 2.5 jamescasei upward 2.75 25 2.75 25 2.75 25 2.75 25 2.75 25 1.25 2 6 8 2.75 25

Photeros downward P.johnbucki down 6.5 6.5 6.5 6.5 5 6.5 (n=10-15) 3.25 arc 3 9 3 9 3 9 3 9 3 9 1 5.5 fixed rapid P. mcelroyi 1 1 1 1 5 1 (n=10-15) 0.25 to up flashing 3 0 3 0 3 0 3 0 3 0 0.75 2 P. rapid up 0.9 0.9 0.9 (n=5-9) 0.28 gramnicola flashing 2 3 2 3 2 3 1.25 20 Pulse 1 Pulse 2 Pulse 3 Pulse 4 Pulse >5 Trill

Clade Species

Pulse#

Direction

Trill number Trill

Pulseduration Pulseduration Pulseduration Pulseduration Pulseduration Pulseduration

Display pattern pattern Display

Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse Interval Iinterpulse

Interpulse Distance InterpulseDistance InterpulseDistance InterpulseDistance InterpulseDistance InterpulseDistance InterpulseDistance

hori- horizontal MSH 2.98 2.8 2.7 2.15 <38 1.63 zontal and long 0.9 1.6 0.55 1.55 0.5 1.2 0.7 1.2 0.36 1.02 widely MWU up 4.2 3.55 3.3 3.1 5 3.05 11.00 1.89 spaced 2.7 12.2 1.8 12.3 1.3 10 1.55 9.5 1.2 9 0.76 5.6 L- horizontal psammobia 4.77 4.76 5.54 5.01 5 5.04 shape to upward 1.77 10.5 2.98 11.5 3.22 8.5 3.42 4.5 6 4.8 3 4.3 7 3.81 3.2 3.6 8 3 2.6 3.4 9 2.87 2.59 3 vertical ignitula down 4.18 4.21 3.26 2.42 5 1.5 no trill downward 2.66 10 1.86 8 1.7 7.2 0.5 1 hori- oblique and noropsela 2.28 (n=250+) 2.28 zontal long 0.58 2 0.58 2 medium scintilla down 8.4 6.85 4.7 4.71 (n=5-10) 1

length 3.79 7 4.8 3.21 3.04 2 2 1 "H" Group very short micamacula down 5.74 3.56 2.7 (n=3-7) 1 downward 3.2 1.75 1.1 0.75 1 0.5 vertical lucidella up 3.3 2.88 2.17 1.89 5 1.78 (n=7-15) 1 upward 2.91 6 1.75 8.4 1.14 5.7 1.32 4.5 0.9 0.9 obli- oblique up contragula 4 3 2 1.9 5 1.9 (dozens) 1.9 que and long 15 3.5 8 2 6 1.5 5 1 3.5 0.4 0.9 6 1.9 0.6 3 kuna up up long, wide 7.5 4.5 4 42.5 3 3 25 1.5 1.5 16.5 5 1.5 1.5 10.5 (n=7-17) 1.5 1.5 8 short kuna down down 2 4 2 1.5 (n=5-6) 1 downward 3.5 6.5 3 4.5 2 3 1.5 2 zigzag up 4.5 4.5 (n=9-29) 4.5 mizonomma upward 3.5 12.5 3.5 12.5 1 4