Proceedings of the 1st Joint Symposium on the Natural History and Geology of The Bahamas June 12-16, 2015 Conference Organizer: Thomas A. Rothfus Content Editors: Carol L. Landry Dan S. Kjar Production Editors: Lee J. Florea Carol L. Landry Gerace Research Centre San Salvador, Bahamas 2017 Copyright 2017, Gerace Research Centre All rights reserved. No part of this work may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopying, recording, or any data storage or retrieval system without the express written permission of the Gerace Research Centre. ISBN: 978-0-935909-40-1 Cover image - Patch reef near the wall off Grotto Beach (photo by Lee Florea). Do reproductive barriers exist between two closely-related species of Croton that occur in sympatry? Leah Schwechheimer and Carol L. Landry Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, OH 1. Abstract observed visiting a virgin flower deposited at least one pollen grain on that flower's stigma. Croton linearis and Croton discolor are There were no significant differences among common plant species in coastal communities treatments in average fruit set or in the average in The Bahamas. They are distinguished number of seeds produced per fruit, and seeds primarily by leaf morphology, not floral from interspecific crosses germinated. The lack characteristics; however, we found individuals of significant differences among pollination in the community that were intermediate with treatments and evidence that hybrid seeds are respect to leaf morphology. To determine viable suggest that the two species are cross- whether these morphological intermediates compatible. Further, the two species may be could be hybrids produced from crosses hybridizing because the primary floral visitors between the two species, we investigated did not differentiate between the two species or potential reproductive barriers that could their morphological intermediates. separate the species. We observed floral visitors to determine if the two species shared 2. Introduction pollinators or if they were reproductively isolated due to a lack of shared pollinators. Studying the dynamics of pollination within Pollen on insects that visited the plants was communities is necessary for successful removed and identified, and following insect conservation (Kearns et al. 1998). Croton visitation, the stigmas of virgin female flowers linearis Jacq. and Croton discolor Willd. were collected, fixed, and screened for Croton (Euphorbiaceae) are common, closely-related, pollen. To determine whether the species were insect-pollinated plants native to The Bahamas cross-compatible, the female flowers of each (Landry et al. 2014a). They are distinguished species were hand-pollinated in March and primarily by differences in leaf morphology; June 2014; flowers received either conspecific Correll and Correll (1982) describe C. discolor pollen or pollen from the other species. We as “a venose, broad-leaved form of C. linearis”. compared average fruit and seed set, and seed On San Salvador, the distribution of C. discolor germination rates for the four treatments. appears to be more restricted than C. linearis, During 26 hours of observation, a total but they do co-occur at several locations of 11 dipteran and hymenopteran species on the island. In at least one location where were observed on flowers of the two species their distributions overlap, morphologically or their intermediates. Five insect species intermediate individuals were previously were responsible for most visits and did observed (Landry, pers. obs.). We investigated not discriminate between plant species, but potential reproductive barriers between C. of these, only four dipteran species visited linearis and C. discolor to determine whether flowers on both male and female plants: the morphological intermediates could be Chrysanthrax maculipennis, Geron salmonus, hybrid individuals. Ligyra cereberus, and Villa lateralis complex. In order to prevent hybridization between Croton pollen was also found on specimens of species found in the same community, at these insects, and one L. cereberus individual least one reproductive barrier must exist 1st JOINT SYMPOSIUM ON THE NATURAL HISTORY AND GEOLOGY OF THE BAHAMAS 143 SCHWECHHEIMER AND LANDRY REPRODUCTIVE BARRIERS IN CROTON SPECIES (Hopkins 2013). In this study, we investigated reference slides prepared from known samples mechanical, gametic, and post-zygotic barriers. by Landry; the two species have identical Mechanical barriers physically prevent pollen morphologies, so pollen identification reproduction between two species; animal- was restricted to genus. pollinated plant species that do not share To determine whether pollinators were able pollinators are isolated from one another via to deposit pollen on the stigma, we observed mechanical barriers. If gametic or post-zygotic visitors to virgin flowers. Female flower buds barriers exist between the Croton species, were covered with mesh bags until the flowers they would prevent successful fertilization or opened. Bags were removed during watches, the development of viable seeds, respectively and any virgin flowers visited by insects were (Lepais et al. 2013). If strong gametic or post- tagged, re-bagged, and collected at the end of zygotic reproductive barriers exist, we do not the experiment; the insect visitors were also expect interspecific crosses to set fruit or seed. collected when possible. Flowers were carried If these barriers exist but are weak, we expect back to the lab at OSU-Mansfield; the stigmas lower fruit and seed set, and lower frequency were dissected from the flowers, mounted on of seed germination in interspecific crosses slides in decolorized aniline blue, and pollen versus crosses made with conspecific pollen. tubes were visualized using fluorescent Finally, if gametic or post-zygotic barriers do microscopy (Kearns and Inouye 1993). not exist, we expect similar fruit set, seed set, To test for gametic and post-zygotic and germination rates in all treatments. barriers, we hand-pollinated the female flowers of each species with either conspecific pollen 3. Methods or with pollen from the other species. There were four treatments: D x D, D x L, L x D, We distinguished the two species and their and L x L (female x male crosses; D = discolor, intermediates by leaf shape and an estimate L = linearis). All open flowers in the same of the leaf width-to-length (W : L) ratio of inflorescence were treated with pollen from the each individual. Plants were categorized as C. same source. Following pollination, the flowers linearis if the leaves were linear and the W : L were covered with mesh bags to exclude insect ratio was < 0.25, and as C. discolor if the leaves visitors and to protect the fruits and seeds until were oblong to elliptic and the W : L ratio was maturation. Mature fruits and seeds from > 0.33; intermediates were “narrowly oblong” flowers pollinated in March were collected or “widely linear” and had intermediate W : L in June, and those from flowers pollinated in ratios. June were collected in December. One-way We made pollinator observations on all ANOVA was used to test for differences in fruit plants in flower that were growing along a 1 km and seed set among treatments. The seeds were transect. For each plant observed, we recorded carried back to OSU-Mansfield, where they the plant category (as defined above), plant were planted in cactus blend potting mix and gender, and all floral visitors. Insects collected allowed to germinate in a growth chamber. while visiting the plants were sacrificed and their pollen loads were removed and transported 4. Results to the lab at OSU-Mansfield. Pollen was clarified via acetolysis and bleached with KOH During March and June, 68 insects representing before mounting in basic fuschin jelly (Kearns 11 species visited Croton flowers during and Inouye 1993). Pollen was also removed approximately 26 hours of observation time from insect specimens that Landry and Elliott (Table 1). Five insect species were responsible had previously collected on the two Croton for most visits and did not discriminate among species. Pollen species were identified using plant categories, but of these, only four dipterans 144 1st JOINT SYMPOSIUM ON THE NATURAL HISTORY AND GEOLOGY OF THE BAHAMAS SCHWECHHEIMER AND LANDRY REPRODUCTIVE BARRIERS IN CROTON SPECIES Table 1. Insect visitors to male and female C. linearis , C. discolor, and their intermediates, and presence or absence of Croton pollen on insect specimens. * = pollen loads not investigated; ** = pollen reported in nest cells (Landry et al. 2014b). Pollen present # Visitors to male and female plants (M / F) Insect species (# specimens) linearis intermediate discolor Diptera Bombyliidae Chrysanthrax maculipennis Yes (4)1 / 20 / 11 / 0 Scarbrough & Davidson Geron salmonus Scarbrough Yes (3)3 / 11 / 12 / 0 & Davidson Ligyra cereberus (F.) Yes (4)4 / 15 / 210 / 4 Villa lateralis complex (Say)Yes (2)0 / 01 / 00 / 1 Syrphidae Ocyptamus cylindricus (F.) No (1)— —— Palpada albifrons (Wied.)Yes (4)0 / 00 / 01 / 0 Hyme noptera Apidae Xylocopa cubaecola Lucas* 0 / 00 / 01 / 0 Crabronidae Microbembex sp. Yes (1)— —— Eumenidae Pachodynerus scrupeus *0 / 00 / 13 / 1 Zavattari Megachilidae Megachile alleni Mitchell Yes**2 / 03 / 010 / 0 Scoliidae Campsomeris t. trifasciata *0 / 00 / 01 / 0 Fabr. Sphecidae Stictia signata (L.) No (1)1 / 00 / 01 / 0 Vespidae Polistes bahamensis Beq. & *0 / 01 / 00 / 1 Salt Total number of insects2011 / 4 11 / 530 / 7 1st JOINT SYMPOSIUM ON THE NATURAL HISTORY AND GEOLOGY OF THE BAHAMAS 145 SCHWECHHEIMER AND LANDRY REPRODUCTIVE BARRIERS IN CROTON SPECIES 100 N = 3; n = 19 N = 3; n = 10 N = 7; n = 17 N = 5; n = 14 90 80 t se 70 t ui 60 Fr 50 % e 40 ag er 30 Av 20 10 0 D x DD x LL x DL x L Treatment Figure 1. Average percent fruit set for hand-pollinated crosses between C. discolor individuals (D x D), C. linearis individuals (L x L), and reciprocal crosses between C. discolor and C. linearis (D x L and L x D), with SE bars.