Journal of Biogeography (J. Biogeogr.) (2016) 43, 1717–1727
ORIGINAL Incipient radiation versus multiple ARTICLE origins of the Galapagos Croton scouleri (Euphorbiaceae) Beatriz Rumeu1,2*, Pablo Vargas1 and Ricarda Riina1
1Real Jard ın Bot anico (CSIC-RJB), Madrid, ABSTRACT Spain, 2Centre for Functional Ecology, Aim Island radiations imply the emergence of numerous species in a short Department of Life Sciences, University of period of time. Downscaling at the infraspecific level, considerable differentia- Coimbra, Coimbra, Portugal tion among populations can be a sign of ‘incipient radiation’. However, this process remains largely unexplored. We focus on one of the most outstanding cases of infraspecific morphological variation in the Gal apagos flora. Our hypothesis is that the phenotypic variation of Croton scouleri is a sign of incipi- ent radiation, in which a single colonization has generated new lineages with considerable morphological differentiation.
Location The Gal apagos Islands and Neotropics. Methods One hundred and forty-four nuclear ribosomal DNA (ITS) and plastid trnL-F sequences of Croton sect. Adenophylli were used to test the hypothesis of a single ancestry (monophyly) of C. scouleri using a phylogenetic approach. Sequence data were analysed using Bayesian inference (BI) and max- imum likelihood (ML). A complementary phylogeographical analysis of C. scouleri and phylogenetically related species was also performed using 123 plastid sequences (trnL-F, petL-psbE, trnH-psbA) in search for common ances- try of Gal apagos lineages. Results The phylogenetic approach revealed that the closest relatives of C. scouleri were C. alnifolius, C. pavonis and C. rivinifolius. However, we failed to support monophyly of C. scouleri populations. Despite finding numerous haplotypes (14 polymorphic sequences/9 substitution-based sequences), their distribution across Croton species prevented us from inferring common ances- try for C. scouleri. The phylogeographical reconstruction revealed multiple lin- eages related to the origin of C. scouleri. Main conclusions Lack of monophyly likely indicates that an incipient radia- tion from a single ancestor does not account for the striking infraspecific phe- notypic variation in C. scouleri. This morphological diversity could be explained by recurrent biogeographical connections between Gal apagos and the mainland, involving multiple colonizations to the islands from the continent rather than back colonizations from the islands to the mainland. Morphologi- cal, reproductive, geographical and ecological evidence better support the sce- nario of recurrent colonizations from the continent in different periods of time. *Correspondence: Beatriz Rumeu, Centre for Functional Ecology, Department of Life Keywords Sciences, Calcßada Martim de Freitas, University Euphorbiaceae, ITS, leaf morphotypes, oceanic islands, petL-psbE, phylogeny, of Coimbra, 3000-456 Coimbra, Portugal. trnH-psbA trnL-F E-mail: [email protected] phylogeography, plastid DNA, ,
ª 2016 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1717 doi:10.1111/jbi.12753 B. Rumeu et al.
geographical correlation (Wiggins & Porter, 1971). These INTRODUCTION characteristics make C. scouleri a good candidate to test the Oceanic islands harbour a great deal of evolutionary radia- hypothesis of incipient radiation on oceanic islands. tions. Characteristics such as isolation, niche availability, The endemic status of C. scouleri in the Gal apagos involves topographic complexity or habitat heterogeneity of many the assumption that all its morphological variation results archipelagos favour diversification and adaptive evolution from a single ancestor that evolved within the archipelago. (Paulay, 1994). Therefore, some of the most striking plant However, this assumption has not been tested by any molec- and animal radiations documented to date have been found ular phylogenetic study, and thus the origin of the species on islands. Among the most significant examples are the remains unclear. Our working hypothesis is that the enor- Hawaiian honeycreepers (Freed et al., 1987) and lobeliads mous phenotypic variation in C. scouleri reflects an incipient (Givnish et al., 2009), the Macaronesian Echium L. (Garc ıa- radiation from a single ancestor. The considerable isolation Maroto et al., 2009) and Aeonium Webb & Berthel. (Jor- of the Gal apagos archipelago (c. 1000-km west of South gensen & Olesen, 2001), and the Gal apagos Scalesia Arn. America) and the absence of diaspore specialization in Cro- (Blaschke & Sanders, 2009) and finches (Grant & Grant, ton for long-distance dispersal also points towards the 2008). hypothesis of a single colonization event (see Vargas et al., The process of evolutionary radiation involves the emer- 2012). Studies on phylogenetic relationships have helped gence of numerous lineages and species from a common identify single versus multiple origins of plants on oceanic ancestor in a short period of time (Schluter, 2000). However, islands based on monophyletic versus polyphyletic groups at the infraspecific level, considerable morphological differen- (see Silvertown, 2004). In this study, we test the monophyly tiation can be a sign of ‘incipient radiation’, where popula- of the Gal apagos lineages of C. scouleri to establish single ori- tions with derived characters represent ‘infant’ lineages over gin from the continent. To this end, a large sample of Croton the course of radiation (Coyne & Orr, 2004; Balao et al., species from section Adenophylli from the Neotropics was 2010). Incipient or not, the first step for the proposal of any used to perform phylogenetic analyses. In addition, a com- radiation is to establish that the current diversification is a plementary phylogeographical approach was explored by consequence of a single origin (Schluter, 2000). In other reconstructing ancestral haplotypes and lineage relationships words, rapid differentiation associated with radiation needs within the Gal apagos Islands. Genealogical relationships help to be preceded by colonization of a single lineage. Knowledge interpret incipient differentiation of insular and continental of the number of colonizations helps determine whether the morphotypes. range of morphological and ecological differentiation among species arose from single or multiple lineages. The same is MATERIALS AND METHODS true at a lower taxonomic level. Incipient radiations have been seldom documented and there is a lack of molecular Study species studies aimed at gaining a deeper insight into significant divergence of populations on islands. Croton scouleri has not been included in any molecular Croton scouleri Hook. f. (Euphorbiaceae) is the only spe- phylogeny; however, it was classified in the Neotropical cies on the Gal apagos Islands belonging to the genus Croton Croton sect. Adenophylli on basis of morphological charac- L., one of the largest monophyletic genera within angios- ters (van Ee et al., 2011). The distribution of C. scouleri in perms (over 1200 spp., van Ee et al., 2011). This species is the volcanic archipelago of Gal apagos encompasses 12 of endemic to the archipelago (Wiggins & Porter, 1971) and the 13 main islands (> 10 km2), being only absent from thus is interpreted as the result of a single colonization from the island of Baltra (Fig. 1). It is an abundant arborescent the Americas (Svenson, 1946b). Most Croton fruits are explo- shrub with a wide habitat range, occurring both in the sive capsules with no adaptations for long-distance dispersal. arid lowlands and moist uplands. Within sect. Adenophylli Nevertheless, C. scouleri is an abundant arborescent shrub (223 species), the narrow-leaved shape is not common and widely distributed across the Gal apagos archipelago (Fig. 1). it is only found in a few species (e.g. C. sagraeanus Morphological variation within this species offers one of the Mull.Arg.€ from Cuba, C. linearis Jacq. from Bahamas, Flor- most outstanding cases of the Gal apagos flora as revealed by ida, Cuba and Jamaica), including the C. scouleri on the its nine taxonomic varieties, of which four had been origi- Gal apagos Islands [C. scouleri f. macraei (Hook. f.) G. L. nally described at the species level (see Wiggins & Porter, Webster, see Appendix S1b]. Croton scouleri was considered 1971). Indeed, variation in plant habit and especially leaf to be exclusively dioecious (Wiggins & Porter, 1971), shape of C. scouleri, led Hamann (1979) to speculate about despite the observation that most of the species of the an ‘early’ speciation within the archipelago. For instance, C. genus Croton are monoecious (van Ee et al., 2011). How- scouleri displays strong extremes in leaf shape, such as broad- ever, monoecy has also been recorded in few individuals leaved forms (see Appendix S1a in Supporting Information) of C. scouleri on the islands of Genovesa, Isabela and and narrow-leaved forms (see Appendix S1b). A wide span Espanola,~ with a frequency of 8–15% of monoecious indi- of intermediate or elliptic-leaved forms between these two viduals in the populations studied (Mauchamp, 1997) (see morphotypes are also frequent (Fig. 2), but show little also Fig. 2d).
1718 Journal of Biogeography 43, 1717–1727 ª 2016 John Wiley & Sons Ltd Origin of the Galapagos Croton scouleri
VENEZUELA ECUADOR COLOMBIA
GALÁPAGOS
BRASIL PINTA PERU (2) BOLIVIA MARCHENA (2) GENOVESA (1)
0° SANTIAGO
FERNANDINA (1) BALTRA
PINZÓN (1)
SANTA CRUZ (8) SANTA FÉ 1° S (1) SAN CRISTÓBAL ISABELA (4)
025 50 100 km FLOREANA (3) ESPAÑOLA (2)
92° W 91° W 90° W 89° W
Figure 1 Map of the Gal apagos archipelago, showing its global location (top-right) and the main islands (> 10 km2). Croton scouleri is distributed in all the main islands except from Baltra. The number of samples sequenced per island is indicated between brackets.
addition, C. caracasanus Pittier (sect. Corylocroton) and C. Sampling and sequencing sampatik Mull.€ Arg. (sect. Sampatik) were used as the out- Plant material was collected in the field and from herbarium group in the phylogeny. Total genomic DNA was isolated specimens [C, HUEFS, LPB, MA, MICH, MO, MU, QCNE, using the DNeasy Plant Mini-Kit (Qiagen Inc., Carlsbad, CA, U, USM, WIS; herbarium acronyms follow Thiers (2015)]. USA) following the manufacturer’s recommended protocols. Several sequences were also obtained from Riina et al. (2009) The internal transcribed spacer region (ITS) of the nuclear (see Appendix S2 for details of the sample origins). Within ribosomal DNA and the plastid spacer trnL-F have proven to our study group we selected representatives of different be useful markers to infer phylogenetic relationships within islands and leaf morphotypes. We included 25 samples of the genus Croton (Berry et al., 2005; Riina et al., 2009). C. scouleri covering much of its morphological variation and Therefore, we chose these two DNA regions in our phyloge- distribution in the Gal apagos Islands (10 out of the 12 netic approach. The ITS region was amplified using the pri- islands of occurrence), and 47 samples of Croton sect. Adeno- mers ITS-1 (Urbatsch et al., 2000) and ITS-4 (White et al., phylli representing most of the morphological variation and 1990). The plastid trnL intron and trnL-F intergenic spacer geographical distribution in the Neotropics (Mexico-Central were amplified using primers ‘c’ and ‘d’, and ‘e’ and ‘f’ America, Caribbean, eastern Brazil, southern South America, respectively (Taberlet et al., 1991). After 2 min pre-treatment Andes). Our sample selection benefited from an ongoing at 94–95 °C, polymerase chain reaction (PCR) conditions phylogenetic analysis based on DNA sequences of sect. were: 30 cycles of 1 min at 95 °C, 2 min at 55–60 °C and Adenophylli (Riina et al., unpubl. data), which helped to 1–2 min at 72 °C; cycles were followed by a final extension infer closely-related taxa. From these 47 samples, 12 were of at 72 °C for 10 min. A volume of 1 lL of bovine serum C. rivinifolius Kunth, an endemic species from Ecuador to albumin at 1 mg mL 1 was included in each 25 lL reaction which C. scouleri was originally ascribed (Svenson, 1946a). In to improve the efficiency of the amplification. PCR products
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(a) (b) (c)
(d)
Figure 2 Morphological variation of Croton scouleri in leaf shape and inflorescence. Scale bar, 1 cm. (a) Broad-leaved morphotype; (b) Elliptic-leaved morphotype; (c) Narrow-leaved morphotype; (d) Monoecious inflorescence at Punta Espejo, Marchena Island. Female flowers are located at the base of the inflorescence axis, whereas male flowers are at more distal positions. Photo (d): P. Vargas. were sequenced using an ABI Prism H 3730xi DNA sequen- assembled a matrix including samples of C. scouleri and their cer at the Macrogen Institute (Macrogen Co., Seoul, Korea). closest mainland relatives based on the phylogenetic analysis. Sequences were aligned, and manually adjusted, using Mafft PCR protocols and sequencing followed the above descrip- 6.814b (Katoh et al., 2002) implemented in the Geneious tion. All new sequences were deposited in GenBank (see 6.1.7 software (Drummond et al., 2011). Appendix S2 for accession numbers). To further explore the origin of C. scouleri variation, we performed a phylogeographical analysis in search for com- Phylogeny reconstruction mon ancestry. First, a pilot study based on three to eight individuals from different islands and 19 plastid DNA Phylogenetic relationships among C. scouleri and its sect. regions was done (Taberlet et al., 1991; Hamilton, 1999; Adenophylli relatives were explored using maximum likeli- Shaw et al., 2005, 2007). Secondly, we chose the following hood (ML) and Bayesian inference (BI) analyses. Prior to three DNA regions as a result of the variability and quality these analyses, the simplest model of sequence evolution that of the amplifications obtained from herbarium samples: best fits each sequence dataset was determined using the trnL-F (Taberlet et al., 1991), trnH-psbA (Hamilton, 1999) Akaike information criterion in jModeltest 2.1.3 (Darriba and petL-psbE (Shaw et al., 2007). For the petL-psbE region, et al., 2012). For BI, MrBayes 3.2.2 (Ronquist & Huelsen- one internal primer (50-CATCGAATATTCTTTACTACTGG- beck, 2003) was used to conduct an exploratory analysis on 30) was herein designed to facilitate the amplification of a the ITS, trnL-F and combined ITS+trnL-F matrices. For each short fragment because of failure in the amplification of the DNA region, four chains were run for five million genera- complete region. For this phylogeographical approach, we tions fitting the optimal models of substitution to each
1720 Journal of Biogeography 43, 1717–1727 ª 2016 John Wiley & Sons Ltd Origin of the Galapagos Croton scouleri molecular partition (SYM+G for ITS, and GTR+G for RESULTS trnL-F). The resulting ITS and trnL-F trees were visually inspected to check for congruence. There was no incongru- Sequence variation and phylogeny ence between the ITS and trnL-F phylogenies, so the concate- nated dataset was analysed. The final BI analysis of the The aligned length of the combined ITS and trnL-F sequences combined dataset was conducted with each model substitu- was 1710 bp (621 bp for ITS; 1089 for trnL-F). There were tion fitted to each molecular partition. Four chains were run 163 variable sites for the ITS and 123 for the trnL-F datasets. twice for 15 million generations with a sample frequency of The ITS alignment had no missing characters, whereas the 1000, and discarding the first 25% generations as burn-in. trnL-F alignment had 3.5% missing characters. Chain convergence was assessed with Tracer 1.5 (Rambaut The two phylogenetic analyses (BI and ML) of Croton sect. & Drummond, 2009), and a 50% majority rule consensus Adenophylli revealed that the closest relatives of C. scouleri tree was calculated to estimate the BI phylogeny. were C. alnifolius Lam. (Ecuador, Peru), C. pavonis Mull.Arg.€ The ML analysis was performed using RAxML (Sta- (Ecuador, Peru) and C. rivinifolius (Ecuador) (Fig. 3). The matakis, 2006) as implemented in raxmlGUI 1.31 (Silvestro four species formed a well-supported clade (Bayesian poste- & Michalak, 2012) with the alignment divided into two par- rior probability = 1; maximum likelihood bootstrap titions (ITS+trnL-F) and a unique GTR*GAMMA model of value = 100). However, this clade had low internal resolution evolution assigned to each partition. Branch support was because the two DNA regions used did not provide enough assessed by performing a thorough bootstrap analysis (1000 variation (two substitutions in ITS; three substitutions in bootstrap replicates) with 100 runs. trnL-F) to infer species relationships and monophyly of C. scouleri populations. Phylogeographical approach Haplotype analysis of C. scouleri and closest Plastid markers are known to be one of the most reliable for relatives inferring plant colonization by seed dispersal (e.g. Guzm an & Vargas, 2009; Fern andez-Mazuecos & Vargas, 2011). The Molecular variation of the ITS sequences in C. scouleri and ptDNA genome is structurally stable, haploid, non-recombi- its closest mainland relatives was defined only by three ribo- nant and maternally inherited in most angiosperms (Birky, types as a result of four substitutions (see Appendix S3). The 2001; Avise, 2009), including species within Euphorbiaceae most common ribotype (R2) was shared by C. scouleri, (Corriveau & Coleman, 1988). The three ptDNA regions C. alnifolius, C. pavonis and C. rivinifolius. Ribotypes R1 and (trnL-F, trnH-psbA and petL-psbE) were concatenated in order R3 were displayed by one sample each of C. alnifolius and to detect polymorphism and reconstruct the ancestral geno- C. scouleri respectively. type (haplotype) of C. scouleri (Birky, 2001). The resulting Variation in the combined ptDNA dataset (trnL-F, petL- dataset was analysed through a statistical parsimony algorithm psbE, trnH-psbA) for C. scouleri and relatives was the result (Templeton et al., 1992), as implemented in tcs 1.21 (Cle- of eight substitutions and four gaps, distributed as follows: ment et al., 2000), to infer genealogical relationships among in trnL-F, three substitutions and one gap; in petL-psbE, haplotypes. The maximum number of differences resulting three substitutions and one gap; in trnH-psbA, two substitu- from single substitutions among haplotypes was calculated tions and two gaps. The analysis of the three combined with 95% confidence limits, and initially treating gaps as miss- ptDNA regions treating gaps as missing data yielded nine ing data. In addition, two more analyses of haplotypic diver- haplotypes of C. scouleri and the three closest mainland rela- sity were performed: (1) by treating gaps as the fifth state, and tives. Haplotype relationships depicted a single network with (2) using ribotypes from the ITS region as a separate dataset. no loops (Fig. 4a). Eight of the nine haplotypes in Fig. 4(a) were found in C. scouleri, and seven were exclusive of this species (1, 3, 4, 5, 6, 7, 8). In contrast, the other three spe- Distribution of leaf morphotypes cies in the clade harboured only two haplotypes (2, 9). Croton scouleri has two main types of leaf shape: broad- Five more haplotypes were detected when considering gaps leaved (Fig. 2a & see Appendix S1a) and narrow-leaved as a fifth state (Fig. 4b). In this case, four missing haplotypes (Fig. 2c & see Appendix S1b) forms. To infer the frequency and one loop were also inferred. Croton scouleri harboured of each leaf morphotype in C. scouleri, we inspected a total 11 haplotypes, nine being private (1, 3, 4, 5, 6, 9, 11, 12, 13), of 107 herbarium specimens representing populations of the whereas C. alnifolius, C. pavonis and C. rivinifolius displayed 12 islands. However, because some herbarium specimens dis- five haplotypes (2, 7, 8 10, 14). From these, two haplotypes played intermediate leaf forms (elliptic), we established three (7, 14) were exclusive of C. rivinifolius. categories for the character leaf morphotype (Fig. 2a–c) and In both haplotype networks (Fig. 4a,b), haplotype 2 was all specimens were visually classified accordingly. The the most frequent and widely distributed, and it was shared frequency of each leaf morphotype has not been previ- by the four species of the ingroup clade. These genealogical ously studied for C. scouleri and this study is considered reconstructions did not provide sufficient resolution to exploratory. precisely infer the origin of all the lineages of C. scouleri.
Journal of Biogeography 43, 1717–1727 1721 ª 2016 John Wiley & Sons Ltd B. Rumeu et al.
02. C. alnifolius (Peru) 01. C. alnifolius (Peru) 03. C. alnifolius (Ecuador) 04. C. pavonis (Ecuador) 09. C. rivinifolius (Ecuador) 06. C. rivinifolius (Ecuador) 08. C. rivinifolius (Ecuador) 05. C. rivinifolius (Ecuador) 16. C. rivinifolius (Ecuador) 10. C. rivinifolius (Ecuador) 13. C. rivinifolius (Ecuador) 11. C. rivinifolius (Ecuador) 15. C. rivinifolius (Ecuador) 12. C. rivinifolius (Ecuador) 07. C. rivinifolius (Ecuador) 14. C. rivinifolius (Ecuador) 24. C. scouleri (Pinzón) 38. C. scouleri (Santa Fé) 40. C. scouleri (Española) 25. C. scouleri (Marchena) 17. C. scouleri (Fernandina) 18. C. scouleri (Isabela) 21. C. scouleri (Isabela) 32. C. scouleri (Santa Cruz) 37. C. scouleri (Floreana) 33. C. scouleri (Santa Cruz) 39. C. scouleri (Genovesa) 28. C. scouleri (Santa Cruz) 31. C. scouleri (Santa Cruz) 19. C. scouleri (Isabela) 23. C. scouleri (Pinta) 29. C. scouleri (Santa Cruz) 22. C. scouleri (Pinta) 27. C. scouleri (Santa Cruz) 41. C. scouleri (Española) 30. C. scouleri (Santa Cruz) 26. C. scouleri (Marchena) 20. C. scouleri (Isabela) 34. C. scouleri (Santa Cruz) 36. C. scouleri (Floreana) 35. C. scouleri (Floreana) 55. C. aff. ferrugineus (Peru) 56. C. aff. ferrugineus (Peru) 71. C. sp (Peru) 72. C. stenosepalus (Peru) 44. C. adipatus (Ecuador) 74. C. thurifer (Peru) 65. C. pungens (Venezuela) 67. C. ruizianus (Peru) 43. C. abutiloides (Ecuador) 45. C. aequatoris (Ecuador) 47. C. chilensis (Chile) 57. C. gracilipes (Bolivia) 42. C. abutilifolius (Bolivia) 68. C. saltensis (Argentina) 70. C. sarcopetalus (Argentina) 63. C. menthodorus (Ecuador) 61. C. menthodorus (Ecuador) 59. C. menthodorus (Ecuador) 64. C. menthodorus (Ecuador) 62. C. menthodorus (Ecuador) 60. C. menthodorus (Ecuador) 51. C. curiosus (Argentina) 58. C. linearis (Bahamas) 52. C. discolor (Puerto Rico) 53. C. echioideus (Brazil) 48. C. conduplicatus (Paraguay) 50. C. cf. conduplicatus (Bolivia) 49. C. conduplicatus (Brazil) 73. C. suberosus (Mexico) 66. C. roxanae (Mexico) 54. C. emporiorum (Bolivia) 46. C. caracasanus (Venezuela) 69. C. sampatik (Peru)
0.2
Figure 3 Fifty per cent majority-rule consensus tree obtained from the Bayesian phylogenetic analysis of ITS and trnL-F sequences of Croton sect. Adenophylli. Black and grey squares above the nodes represent Bayesian posterior probabilities ≥ 95% and maximum likelihood bootstrap values ≥ 75%, respectively. Sample provenance (country) is indicated within brackets after taxa names, except for C. scouleri (grey shaded), for which each Gal apagos island is indicated.
However, it is notable that in one analysis haplotype 9 Leaf and inflorescence variation appears unrelated to the other 10 haplotypes of C. scouleri (see Fig. 4b), needing a minimum of four steps to be con- Visual inspection of 107 herbarium specimens yielded differ- nected to haplotype 2. The absence of loops in the clade ent frequencies of occurrence of the three leaf morphotypes. including these haplotypes suggests lack of homoplasy, two The most abundant morphotype was broad-leaved (62.6%), different lineages in the Gal apagos and thus, rejecting the followed by the narrow-leaved (23.4%) and the intermediate hypothesis of a single origin of C. scouleri. elliptic-leaved (14.0%).
1722 Journal of Biogeography 43, 1717–1727 ª 2016 John Wiley & Sons Ltd Origin of the Galapagos Croton scouleri
(a) (b) 9 7 8 6 6 7 5 5 8 11 2 2 4 12 4 2 9 3 10 13 1 3 14 1
OUT OUT
INGROUP a b a b HAPLOTYPE HAPLOTYPE HAPLOTYPE HAPLOTYPE 01. C. alnifolius - Peru 2 2 22. C. scouleri - Pinta 2 2 02. C. alnifolius - Peru 2 2 23. C. scouleri - Pinta 2 2 03. C. alnifolius - Ecuador 2 8 24. C. scouleri - Pinzón 8 6 04. C. pavonis - Ecuador 2 8 25. C. scouleri - Marchena 2 2 05. C. rivinifolius - Ecuador 2 2 26. C. scouleri - Marchena 2 2 06. C. rivinifolius - Ecuador 2 2 27. C. scouleri - Santa Cruz 2 2 07. C. rivinifolius - Ecuador 2 7 28. C. scouleri - Santa Cruz 2 2 08. C. rivinifolius - Ecuador 2 10 29. C. scouleri - Santa Cruz 3 9 09. C. rivinifolius - Ecuador 2 2 30. C. scouleri - Santa Cruz 2 4 10. C. rivinifolius - Ecuador 9 14 31. C. scouleri - Santa Cruz 4 13 11. C. rivinifolius - Ecuador 2 2 32. C. scouleri - Santa Cruz 2 2 12. C. rivinifolius - Ecuador 2 7 33. C. scouleri - Santa Cruz 8 6 13. C. rivinifolius - Ecuador 2 2 34. C. scouleri - Santa Cruz 5 5 14. C. rivinifolius - Ecuador 2 2 35. C. scouleri - Floreana 7 11 15. C. rivinifolius - Ecuador 2 2 36. C. scouleri - Floreana 6 12 16. C. rivinifolius - Ecuador 2 2 37. C. scouleri - Floreana 2 2 17. C. scouleri - Fernandina 2 2 38. C. scouleri - Santa Fe 2 2 18. C. scouleri - Isabela 2 10 39. C. scouleri - Genovesa 2 2 19. C. scouleri - Isabela 2 2 40. C. scouleri - Española 2 2 20. C. scouleri - Isabela 2 1 41. C. scouleri - Española 2 2 21. C. scouleri - Isabela 1 3
Figure 4 Statistical parsimony networks of Croton scouleri and its closest relatives (ingroup, Fig. 3) based on ptDNA haplotypes (trnL-F, petL-psbE and trnH-psbA). (a) Substitution-based haplotype network (treating gaps as missing data). (b) Polymorphic haplotype network (treating gaps as fifth state). In both networks, each haplotype is represented by a colour and a number. Lines represent single nucleotide substitutions; dots indicate absent haplotypes (extinct or not found). Haplotype circle size is proportional to frequency. The two haplotypes (a, b) for each sample are indicated in the inset. Sample provenance (country) is indicated after taxa names, except for C. scouleri, for which each Gal apagos island is shown. See Appendix S2 for specific information of each sample.
Our search for sexual expression of C. scouleri flowers DISCUSSION across the main islands (Espanola,~ Fernandina, Floreana, Genovesa, Isabela, Marchena, Pinta, Pinzon, San Cristobal, Island biota has provided some of the most informative cases Santa Cruz, Santa F e and Santiago) supports dioecy as the of radiations worldwide, and early stages of radiations have predominant reproductive system. However, monoecy has been interpreted as incipient diversification within lineages been recently found on the island of Marchena, where inflo- (e.g. Vijverberg et al., 2000; McGlaughlin & Friar, 2011; Stacy rescences with male and female flowers were observed on five et al., 2014). Nevertheless, in light of the phylogenetic and individuals (A. Traveset, pers. obs., Fig. 2d). phylogeographical results, the pronounced morphological
Journal of Biogeography 43, 1717–1727 1723 ª 2016 John Wiley & Sons Ltd B. Rumeu et al. variation in the Gal apagos C. scouleri does not seem to be of occurrence) in the Gal apagos populations. This is also the the result of an incipient radiation. Neither the ITS and most common morphotype of the three closest Croton rela- trnL-F phylogeny nor the ptDNA network supported the tives (C. alnifolius, C. pavonis and C. rivinifolius) in the con- monophyletic origin of the species and, consequently, our tinent. The fact that narrow-leaved plants are found only in results suggest that alternative hypotheses to incipient radia- Gal apagos populations (c. 25% of the individuals inspected), tion better explain the infraspecific phenotypic variation together with the high number of haplotypes, indicate rela- found in C. scouleri. tively ancient occurrence and differentiation of Croton on the islands. The great morphological variation in C. scouleri paralleled Continental relatives and morphological the high diversity of plastid haplotypes. In particular, differentiation of C. scouleri C. scouleri sequence variation, with eight of the nine substi- Croton scouleri has been the focus of taxonomic disagreement tution-based haplotypes (Fig. 4a) and 11 of 15 polymorphic as a result of its striking morphological variation. Wiggins & haplotypes (Fig. 4b), outnumbers that of its closest mainland Porter (1971) refer to its variation patterns as ‘nearly intract- relatives (two substitution-based and five polymorphic hap- able to rational taxonomic treatment’. Our DNA sequence lotypes). In other words, most of these haplotypes were pri- analyses support its phylogenetic relationship with C. alni- vate to C. scouleri. Both narrow-leaved and broad-leaved folius, C. pavonis and C. rivinifolius from Ecuador and Peru, individuals showed exclusive haplotypes (see Appendix S2). with which C. scouleri forms a well-supported clade. Some However, any relationships between haplotypes and morpho- morphological characters also link these four species into a types were not evident. A deeper phylogeographical research natural group. They all share stellate trichomes, indumentum of C. scouleri within the Gal apagos archipelago would be density and distribution, petiolar glands, and inflorescence necessary to test any correlation between genetic diversity and flower morphology. Regarding to the inflorescence, how- and morphotypes, especially regarding leaf and seed mor- ever, some differences arise among the four taxa. Dioecious phology. individuals (dominant) coexisting with monoecious plants (less frequently) have been documented for C. scouleri (see Multiple colonizations versus back colonization Mauchamp, 1997 and Fig. 2d in this study). Croton alnifolius and C. rivinifolius are considered dioecious species, although Lack of strong phylogenetic distinctiveness of populations of a case of monoecy in C. rivinifolius has been detected in a C. alnifolius, C. pavonis, C. rivinifolius and C. scouleri was herbarium specimen inspected by us (Riina, Real Jard ın demonstrated by the absence of monophyletic groups within Bot anico (CSIC-RJB), Madrid, pers. obs.). On the other the well-supported clade that comprises these four species. hand, C. pavonis seems consistently monoecious (Riina, pers. Besides, no single common ancestry of the main Gal apagos obs.). The four species also show ecological similarities and haplotype lineages is inferred from the haplotype network differences. Croton scouleri is widely distributed in the archi- analysis. Therefore, the four species may have shared recent pelago of Gal apagos, from arid lowlands (where it can be geographical and evolutionary histories. Once we failed to dominant) to moist uplands (0–1600 m a.s.l.) (Hamann, find support for single origin of C. scouleri, and thus incipi- 1979). In the mainland, C. pavonis occurs mainly from ent radiation, the question remains as to whether this pattern 1500–2000 m a.s.l. in humid montane forests (Jørgensen & of morphological variation is exclusively the result of multi- Leon-Y anez, 1999). Croton rivinifolius forms part of the ple biogeographical connections between Gal apagos and the coastal vegetation (0–200 m a.s.l.), and Croton alnifolius mainland. In other words, biogeographical relationships occurs in the Peruvian coastal desert, where the vegetation is among Croton lineages in the Gal apagos may be the result of largely restricted to the fog-zone or lomas (small hills) two non-mutually exclusive origins: (1) multiple coloniza- between 200–800 m (Dillon et al., 2011). tions from the continent, or (2) back colonization from the Taxonomic proximity of C. scouleri and C. rivinifolius had islands to the mainland. already been stressed by Svenson (1946a), who included the Oceanic islands are difficult to be colonized by most former in the latter based on their morphological similarities. organisms. As a result, the majority of plant lineages on such However, Webster (1970) and Wiggins & Porter (1971) islands are monophyletic, which indicates that they origi- re-instated C. scouleri as a distinct and endemic species to nated from a single colonization event (Gillespie & Clague, the Gal apagos archipelago arguing that the seeds of C. rivini- 2009). However, recent molecular phylogenetic analyses have folius differ from those of C. scouleri in being ‘distinctly provided valuable insights into the relationships of oceanic grooved or ridged instead of pitted’. Indeed, seed and leaf floras and their mainland relatives, showing that multiple morphologies have been used to delimit morphological vari- colonizations of the islands have also occurred in a consider- ation in C. scouleri (Fig. 2a–c & see Appendix S1) into nine able number of genera (Carine et al., 2004). In the Gal apagos taxa by numerous botanists (e.g. J. D. Hooker, N. J. Ander- flora, phylogenetic evidence for multiple origins has already sson, H. K. Svenson, A. N. Stewart). been demonstrated for Darwiniothamnus Harling (Andrus Our inspection of herbarium specimens indicated that the et al., 2009), Cordia L. (Weeks et al., 2010), Cuscuta L. (Ste- broad-leaved form appears to be the most abundant (62.6% fanovi c et al., 2007) and Gossypium L. (Wendel et al., 2009).
1724 Journal of Biogeography 43, 1717–1727 ª 2016 John Wiley & Sons Ltd Origin of the Galapagos Croton scouleri
Multiple colonizations have been also hypothesized for sequences (next generation sequencing data) correlated with c. 25% of the genera of the Gal apagos archipelago (Vargas individual phenotypes (biometric data) would help test the et al., 2012). hypotheses of (1) two or more colonizations to the islands, In contrast, back-colonization to the mainland, and thus (2) leaf morphological differentiation correlated with particu- islands serving as source area for continental taxa, appears to lar genotypes, and (3) whether two different leaf and seed be a less frequent phenomenon. Nevertheless, the island-to- morphotypes indicate differentiation at different times, and continent colonization hypothesis has been proposed in a few thus asynchronous colonization of Gal apagos archipelago. insular groups, such as Aeonium (Crassulaceae) (Mort et al., 2002) and Tolpis Adans. (Park et al., 2001; Moore et al., 2002) ACKNOWLEDGEMENTS from the Canary Islands (see Caujap e-Castells, 2011 for a review with emphasis on the Canary Islands). There is even the This research is framed within a project financed by the Spanish example of Convolvulus L., also from the Canary Islands, which Ministry of Economy and Competitiveness (CGL2012-38624- shows phylogenetic patterns compatible with both multiple C02). R. Riina was supported by two grants from the EU pro- mainland-archipelago colonizations and one case of back colo- gram Synthesys (GB-TAF-2824, NL-TAF-3710). We thank E. nization (Carine et al., 2004). To the best of our knowledge, Cano for technical support. The Charles Darwin Foundation no case of back-colonization has been documented so far in (CDF) and the Gal apagos National Park (Ecuador) provided the flora of the Gal apagos Islands. logistic support. We are grateful to herbaria AAU, C, E, HUEFS, LPB, MA, MICH, MO, NY, POM, QCA, RSA, U, UC and WIS for allowing us access to their collections. Hypothesis of multiple colonizations
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