A molecular phylogeny of the Pacific clade of Cyrtandra (Gesneriaceae) reveals a Fijian origin, recent diversification, and the importance of founder events

Accepted Manuscript A molecular phylogeny of the Pacific clade of Cyrtandra (Gesneriaceae) reveals a Fijian origin, recent diversification, and a history of long-distance dispersal Melissa A. Johnson, John R. Clark, Warren L. Wagner, Lucinda A. McDade PII: DOI: Reference:

S1055-7903(17)30507-9 http://dx.doi.org/10.1016/j.ympev.2017.07.004 YMPEV 5867

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

27 September 2016 1 May 2017 7 July 2017

Please cite this article as: Johnson, M.A., Clark, J.R., Wagner, W.L., McDade, L.A., A molecular phylogeny of the Pacific clade of Cyrtandra (Gesneriaceae) reveals a Fijian origin, recent diversification, and a history of longdistance dispersal, Molecular Phylogenetics and Evolution (2017), doi: http://dx.doi.org/10.1016/j.ympev. 2017.07.004

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A molecular phylogeny of the Pacific clade of Cyrtandra (Gesneriaceae) reveals a Fijian origin, recent diversification, and a history of long-distance dispersal Melissa A. Johnsona,*, John R. Clarkb, Warren L. Wagnerc, Lucinda A. McDadea a

Rancho Santa Ana Botanic Garden, Claremont Graduate University, 1500 North College

Avenue, Claremont, CA 91711, USA. b

Center for Plant Conservation at San Diego Zoo Global, National Headquarters, 15600 San

Pasqual Valley Road, Escondido, CA 92027, USA. c

Department of Botany, MRC 166, Smithsonian Institution, PO Box 37012, Washington, DC

20013-7012, USA.

*Corresponding author. Present address: USDA-Agricultural Research Service, Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center, 64 Nowelo Street, Hilo, HI 96720, USA. Email addresses: [email protected], [email protected] (M.A. Johnson)

 

ͳ

ͳ

ABSTRACT Cyrtandra (Gesneriaceae) is among the largest genera of flowering plants in the remote

ʹ ͵

oceanic islands of the Pacific, with an estimated 175 species distributed across an area that

Ͷ

extends from the Solomon Islands, east to the Marquesas Islands, and north to the Hawaiian

ͷ

Islands. The vast majority of species are single-island endemics that inhabit upland rainforests.

͸

Although previous molecular phylogenetic studies greatly advanced our understanding of the

͹

diversification of Pacific Cyrtandra, a number of uncertainties remain regarding phylogenetic

ͺ

relationships, divergence times, and biogeographic patterns within this large and widely

ͻ

dispersed group. In the present study, five loci (ITS, ETS, Cyrt1, psbA-trnH, and rpl32-trnL)

ͳͲ

were amplified and sequenced for phylogenetic reconstruction of 121 Cyrtandra taxa. Maximum

ͳͳ

likelihood and Bayesian inference confirmed that C. taviunensis from Fiji is sister to the

ͳʹ

remaining members of the Pacific clade. Dating analyses and ancestral area estimation indicates

ͳ͵

that the Pacific clade of Cyrtandra originated in Fiji during the Miocene ca. 9 mya. All major

ͳͶ

crown lineages within the Pacific clade appeared < 5 mya, coincident with the emergence of

ͳͷ

numerous Pacific islands and a subsequent increase in available habitat. The biogeographic

ͳ͸

history of Cyrtandra in the Pacific has been shaped by extinction, dispersal distance, and founder

ͳ͹

events. Six founder events each from Fiji and Samoa resulted in the broad distribution of

ͳͺ

Cyrtandra seen in the Pacific today. Biogeographic stochastic mapping analyses suggest that

ͳͻ

cladogenesis within Pacific Cyrtandra was exclusively the result of founder events. A mean of

ʹͲ

25 founder events was recovered between Pacific archipelagos, while a mean of 10 founder

ʹͳ

events was recovered within the Hawaiian archipelago.

ʹʹ

Keywords: Ancestral area estimation, Pacific island biogeography, Fiji, founder events,

ʹ͵

Hawaiian Islands, phylogenetic dating

 

ʹ

ʹͶ

1. Introduction Oceanic islands have long been viewed as natural laboratories for the study of

ʹͷ ʹ͸

evolutionary patterns and processes. Across the Pacific Basin, islands vary greatly in age, area,

ʹ͹

elevation and degree of isolation, and their biotas differ in taxonomic composition, origin and

ʹͺ

richness (Keast and Miller, 1996). This array of islands provides an excellent testing ground for

ʹͻ

novel approaches to biogeographical analysis. Research examining the biogeographic history of

͵Ͳ

Pacific plant radiations to date suggests that Malesia, Australia, New Zealand, and New

͵ͳ

Caledonia appear to be significant source areas for remote island taxa (reviewed in Keppel et al.,

͵ʹ

2009). The Americas, Africa, and subarctic regions appear to be the source area for fewer taxa,

͵͵

although there has been a strong contribution from these regions to the Hawaiian flora (Baldwin

͵Ͷ

and Wagner, 2010; Keeley and Funk, 2011). These findings are in contrast to the long held belief

͵ͷ

that Malesia (i.e., Southeast Asia) is by far the most frequent source area for colonists into the

͵͸

Pacific (reviewed in Keppel et al., 2009), an idea mainly attributed to high species diversity

͵͹

within Malesia and shared floristic affinities between Malesia and islands of the Southwest

͵ͺ

Pacific (van Balgooy, 1971).

͵ͻ

There is also increasing evidence that supports long-distance dispersal via wind, ocean

ͶͲ

currents, and birds as a major factor contributing to the establishment of island taxa (Carlquist,

Ͷͳ

1967; reviewed in Nathan, 2006; Gillespie et al., 2012; Le Roux et al., 2014). In contrast, little

Ͷʹ

evidence has been found in support of vicariance mechanisms in shaping species distributions on

Ͷ͵

Pacific islands, and this is limited to some geological evidence of continental fragments drifting

ͶͶ

eastward from New Caledonia to Fiji (reviewed in Keppel et al., 2009). Lastly, while

Ͷͷ

phylogenetic relationships among species are increasingly being used to infer dispersal history

Ͷ͸

among islands, extinction can erase evidence of colonization, particularly in older lineages

 

͵

Ͷ͹

(Emerson, 2002). This can lead to misinterpretation of both source areas and modes of

Ͷͺ

colonization (reviewed in Keppel et al., 2009). To better understand the origins of island taxa, as

Ͷͻ

well as the biogeographic processes involved in creating present-day species distributions,

ͷͲ

further studies are needed that combine molecular phylogenies with geographically

ͷͳ

representative sampling among closely related species complexes. The genus Cyrtandra J.R. & G. Forster (Gesneriaceae) is a diverse group of understory

ͷʹ ͷ͵

plants, with ca. 800 species distributed across Southeast Asia and the Pacific (Atkins et al.,

ͷͶ

2013). Centers of diversity for the group include Borneo (ca. 200 spp.), the Philippines (ca. 150

ͷͷ

spp.), and New Guinea (ca. 120 spp.). The remote volcanic islands of the Pacific are also

ͷ͸

exceptionally species-rich, with ca. 175 Cyrtandra species distributed across an area that extends

ͷ͹

from the Solomon Islands, east to the Marquesas Islands, and north to the Hawaiian Islands

ͷͺ

(Atkins et al., 2013). Species of Cyrtandra are morphologically diverse in habit (small trees,

ͷͻ

shrubs, or vines), flower color (pink, red, purple, yellow, green, white), and fruit type

͸Ͳ

(indehiscent capsule or fleshy berry). However, plants of Pacific species of Cyrtandra are

͸ͳ

predominantly small trees or shrubs with white flowers and white fleshy fruits. Species generally

͸ʹ

inhabit montane to lowland rainforests and occasionally mesic valleys, although several species

͸͵

occur near sea level. Cyrtandra species are diploid (2n = 34; Kiehn, 2005), and evidence for

͸Ͷ

reproductive isolation via postzygotic barriers has been demonstrated for several pairs of taxa in

͸ͷ

the Hawaiian Islands (Johnson et al., 2015). Samuel et al. (1997) were the first to include Pacific Cyrtandra in a molecular

͸͸ ͸͹

phylogenetic analysis; these authors examined relationships among 10 species of Cyrtandra and

͸ͺ

several outgroup taxa using the atpB-rbcL chloroplast (cp) spacer region. Using maximum

͸ͻ

parsimony, their results suggested a paraphyletic Samoan clade and an unresolved relationship

 

Ͷ

͹Ͳ

between Malaysian and Hawaiian taxa. Cronk et al. (2005) later presented a phylogeny based on

͹ͳ

the nuclear ribosomal (nr) ITS region that included 36 Cyrtandra species. Maximum parsimony

͹ʹ

and Bayesian inference supported a single introduction of Cyrtandra to the Pacific from

͹͵

mainland sources. A sister relationship between the single known Taiwanese species, C.

͹Ͷ

umbellifera, and the Pacific clade was also inferred. More recently, several studies by Clark et al.

͹ͷ

(2008, 2009, 2013) greatly increased sampling to include 70 species and sequence data for the

͹͸

nrITS and nrETS regions, as well as the psbA-trnH cp spacer region. Using maximum likelihood

͹͹

and Bayesian inference, the combined results of these analyses support several major findings: 1)

͹ͺ

Southeast Asian species are situated at the base of the tree, forming a grade of several clades, 2)

͹ͻ

C. umbellifera from Taiwan is nested within a clade of species from Southeast Asia, 3) two

ͺͲ

species from the Solomon Islands are supported as sister to the monophyletic Pacific clade, 4)

ͺͳ

the Solomon Islands, Fiji, Samoa, and the Society Islands all host species that are placed in more

ͺʹ

than one clade; in contrast the Marquesas and Hawaiian lineages are monophyletic, and 5) a

ͺ͵

greater Fiji-Samoa region appears to have played a central role in the expansion of the genus

ͺͶ

across the Pacific. Previous molecular phylogenetic studies have greatly advanced our understanding of the

ͺͷ ͺ͸

diversification of Pacific Cyrtandra, but a number of uncertainties remain regarding

ͺ͹

phylogenetic relationships, divergence times, and overarching biogeographic patterns within this

ͺͺ

large and widely dispersed genus. To further advance our understanding beyond previous work,

ͺͻ

we conducted a new and rigorous time-calibrated phylogenetic study for Pacific Cyrtandra based

ͻͲ

on three nuclear and two chloroplast genes, with 82–99% coverage of gene regions for all

ͻͳ

sampled taxa. Our taxon sample was designed to cover all major geographic regions across

ͻʹ

Southeast Asia and the Pacific Basin, with an emphasis on Pacific archipelagos that host marked

 

ͷ

ͻ͵

levels of species diversity. We then use this improved phylogenetic framework for Cyrtandra to

ͻͶ

1) evaluate relationships among Pacific lineages, 2) estimate when and where major lineages of

ͻͷ

Pacific Cyrtandra originated, and 3) infer the biogeographic processes that may have contributed

ͻ͸

to present day distributions. Insights gained from this study will improve our understanding of

ͻ͹

the processes involved in the diversification of plants across the Pacific, particularly in the

ͻͺ

floristically diverse and biogeographically complex South Pacific.

ͻͻ ͳͲͲ

2. Material and Methods

ͳͲͳ

2.1 Taxon sampling We sampled a total of 163 accessions representing 121 taxa in Cyrtandra (Table 1).

ͳͲʹ ͳͲ͵

Species of Aeschynanthus L. and Agalmyla Blume were included as outgroups to root the

ͳͲͶ

phylogeny based on the relationships inferred in Roalson and Roberts (2016). Sampling within

ͳͲͷ

Cyrtandra built on the earlier work of Clark et al. (2008, 2009, 2013), adding 60 species from

ͳͲ͸

across Southeast Asia and the Pacific including Borneo, Sulawesi, Sumatra, Philippines, Papua

ͳͲ͹

New Guinea, Solomon Islands, Vanuatu, Loyalty Islands, Wallis and Futuna, Fiji, Samoa,

ͳͲͺ

Society Islands, Austral Islands, and the Hawaiian Islands (see Table 2 for number of species

ͳͲͻ

sampled by region). Several samples from the Solomon Islands, Vanuatu, Fiji, and the Society

ͳͳͲ

Islands that have not yet been identified to species, as well as five species from Fiji that are in the

ͳͳͳ

process of being described (Johnson, in prep), were included based on morphological and genetic

ͳͳʹ

distinctness. Given that the Fiji-Samoa region was previously identified as a significant

ͳͳ͵

biogeographic interface between Southeast Asia and the Pacific (Clark et al. 2008, 2009), we

ͳͳͶ

focused our sampling efforts in this region of the Pacific to acquire a total of 30 Fijian species (of

ͳͳͷ

an estimated 46) during fieldwork in 2014 and 2015, and a total of 10 Samoan species (of an

 

͸

ͳͳ͸

estimated 20) during fieldwork in 2016. Multiple accessions of each species were included when

ͳͳ͹

possible (one to two accessions for species with restricted distributions, three to four accessions

ͳͳͺ

for more widespread species). Leaf samples were preserved in silica gel and vouchers deposited

ͳͳͻ

at Rancho Santa Ana Botanic Garden (RSA), the South Pacific Regional Herbarium (SUVA),

ͳʹͲ

and the National Tropical Botanical Garden (PTBG). To ensure accurate identification of

ͳʹͳ

species, morphological comparisons were made with all existing species descriptions (Gillett,

ͳʹʹ

1967; Smith, 1991) and herbarium specimens from BISH, GH, K, NY, RSA, SUVA, UC, and

ͳʹ͵

US.

ͳʹͶ

2.2 Molecular methods Genomic DNA was extracted from silica-dried leaf tissue using either Qiagen DNeasy

ͳʹͷ ͳʹ͸

Plant Mini Kits (Qiagen, Valencia, California) or a modified CTAB procedure (Doyle and

ͳʹ͹

Doyle, 1987). The methods of Clark et al. (2008) were followed for amplifying the nrITS and

ͳʹͺ

nrETS regions, as well as cp psbA-trnH. The Cyrt1 low-copy nuclear marker (Cyrtandra

ͳʹͻ

specific, coding for a translationally controlled tumor protein and spanning three exons and two

ͳ͵Ͳ

introns) was amplified following Pillon et al. (2013a). PCR amplification of the rpl32-trnL cp

ͳ͵ͳ

region was carried out in 25 ȝL reactions using 13.75 ȝL of H20, 5.0 ȝL of 5x Go-Taq Flexi

ͳ͵ʹ

Buffer (Promega, Madison, Wisconsin), 1.5 ȝL of 25 mM Mgcl2, 1.0 ȝL of 2.5 mM dNTPs, 0.8

ͳ͵͵

ȝL each of forward (rpl32-F) and reverse (trnL (UAG)) primers, 1.0 ȝL of Bovine-Serum

ͳ͵Ͷ

Albumin (BSA), 0.15 ȝL of Go-Taq Flexi Polymerase, and 1.0 ȝL of DNA template, using the

ͳ͵ͷ

following thermocycler protocol: an initial denaturation of 4 min at 95°C; followed by 35 cycles

ͳ͵͸

of 30 s at 95°C, 1 min at 54°C, 2 min at 72°C; and a final extension of 10 min at 72°C.

ͳ͵͹

Amplification products were visualized on 1% agarose gels, and purified using polyethylene

ͳ͵ͺ

glycol 20% (PEG) precipitation.

 

͹

Purified PCR products were directly sequenced using the PRISM Big Dye 3.1 terminator

ͳ͵ͻ ͳͶͲ

cycle-sequencing reaction (Applied Biosystems, Foster City, California) following the

ͳͶͳ

manufacturer’s protocol. Forward and reverse sequencing reactions were conducted for sequence

ͳͶʹ

confirmation. Cycle sequence products were cleaned using Sephadex G50 AutoSeq columns (GE

ͳͶ͵

Healthcare, Diegem, Belgium), and sequenced on an ABI 3100 genetic analyzer (Applied

ͳͶͶ

Biosystems). Sequence chromatograms were checked for quality, assembled into contigs, and

ͳͶͷ

edited using Geneious v. 9.0.5 (Kearse et al., 2012). Sequences of nrITS, nrETS, and cp psbA-

ͳͶ͸

trnH were downloaded from GenBank for samples used in previous phylogenetic analyses (Clark

ͳͶ͹

et al., 2008, 2009, 2013) when we had silica-dried material of these same specimens available to

ͳͶͺ

us for further sequencing of the nuclear Cyrt1 and cp rpl32-trnL gene regions.

ͳͶͻ

Alignments were inferred using MUSCLE (Edgar, 2004) as implemented in Geneious

ͳͷͲ

(Kearse et al., 2012), followed by manual editing. The identity of each amplified genic region

ͳͷͳ

was validated through BLAST searches in GenBank. A homoplastic 31 bp inversion in the 3' end

ͳͷʹ

of psbA-trnH, and a highly variable AT repeat region (ca. 55 bp) in the 5' end of the same region

ͳͷ͵

were removed from alignments following Clark et al. (2008). Sites with multiple signals were

ͳͷͶ

coded using the IUPAC ambiguity codes.

ͳͷͷ

2.3 Phylogenetic analyses PartitionFinder v. 2.1.1 (Guindon et al., 2010; Lanfear et al., 2016) was used to determine

ͳͷ͸ ͳͷ͹

the appropriate data-partitioning scheme. We used the greedy algorithm (Lanfear et al., 2012)

ͳͷͺ

and the Aikake Information Criterion (AIC) to find the optimal partition scheme from 13

ͳͷͻ

partitions that were determined a priori based on coding and non-coding regions. The best

ͳ͸Ͳ

scoring partition scheme (SI Table S1) was used for Maximum Likelihood (ML) and Bayesian

ͳ͸ͳ

Inference (BI). Model testing, ML, and BI analyses were all conducted using the CIPRES

 

ͺ

ͳ͸ʹ

Science Gateway v. 3.3 (Miller et al. 2010). The best-fit model of nucleotide evolution for each

ͳ͸͵

genic region was selected using the AIC criterion as implemented in jModelTest v. 2.1.6

ͳ͸Ͷ

(Darriba et al., 2012; see SI Table S1). ML analyses were conducted with RAxML v. 8.2.6

ͳ͸ͷ

(Stamatakis, 2006; Stamatakis et al., 2008) using the GTRCAT model of sequence evolution.

ͳ͸͸

The search for the optimal ML tree was done using default search parameters and a rapid

ͳ͸͹

bootstrap analysis of 1000 replicates. Clades with bootstrap support (BS) • 80% were considered

ͳ͸ͺ

well supported. ML analyses were initially conducted on individual genic regions to assess

ͳ͸ͻ

congruence, with areas of conflict determined by examining the placement of individual taxa on

ͳ͹Ͳ

each gene tree. Relationships were considered incongruent if the placement of taxa varied among

ͳ͹ͳ

the individual gene trees and exhibited BS values • 80%. An additional ML analysis was

ͳ͹ʹ

conducted on the concatenated (nuclear + cp) partitioned dataset. Multiple RAxML runs (using a

ͳ͹͵

random starting seed to test the consistency of tree topology and node support) produced

ͳ͹Ͷ

identical results. Bayesian inference analyses were conducted using MrBayes v. 3.2.6 (Ronquist et al.,

ͳ͹ͷ ͳ͹͸

2012). Two independent Bayesian runs with four chains (one cold and three heated) of Markov

ͳ͹͹

Chain Monte Carlo (MCMC) were run for 30 million generations, sampling every 10,000

ͳ͹ͺ

generations. Chain convergence was checked in Tracer v. 1.6 (Rambaut et al., 2014) by

ͳ͹ͻ

examining log likelihood plots, and ensuring that Effective Sample Size (ESS) values were well

ͳͺͲ

above 200. After discarding 25% of the trees as burn-in, a majority rule consensus tree was

ͳͺͳ

constructed using TreeAnnotator v. 2.3.2 (Drummond et al., 2012). Clades with posterior

ͳͺʹ

probability (PP) values • 0.95 were considered well supported. For ML and BI analyses, tree

ͳͺ͵

topology and node support were examined in FigTree v. 1.4.2

ͳͺͶ

(http://tree.bio.ed.ac.uk/software/figtree/).

 

ͻ

ͳͺͷ

2.4 Divergence time estimates

ͳͺ͸

To estimate divergence times we generated a time-calibrated phylogeny using the

ͳͺ͹

partitioned five-gene dataset in BEAST v. 2.4.4 (Drummond et al., 2012), as implemented in the

ͳͺͺ

CIPRES Science Gateway v. 3.3 (Miller et al., 2010). The tree was modeled under a Yule

ͳͺͻ

process using a random starting tree, and an uncorrelated relaxed clock with a lognormal

ͳͻͲ

distribution. Substitution model parameters for each of the eight partitions were based on the

ͳͻͳ

estimated best-fit model of nucleotide evolution. Two independent MCMC analyses were run for

ͳͻʹ

300 million generations each, and sampled every 10,000 generations. Given that there are no

ͳͻ͵

known fossils within the Gesneriaceae family, the phylogeny was constrained using a

ͳͻͶ

combination of geologic ages and secondary calibration points. The geological history of the

ͳͻͷ

Hawaiian Islands is well known, being formed in a linear chronological sequence (with islands

ͳͻ͸

increasing in age towards the WNW) by a hotspot in the Pacific plate. Substrate age for these

ͳͻ͹

volcanic islands has been studied extensively using a number of dating methods, including K–Ar

ͳͻͺ

and Ar–Ar geologic dating techniques (Sharp et al., 1996; Sharp and Renne, 2005; Sherrod et al.,

ͳͻͻ

2007). As such, we chose to use the maximum geologic age of the main Hawaiian Islands as an

ʹͲͲ

age constraint. The Hawaiian Islands calibration point was assigned a lognormal distribution,

ʹͲͳ

with an upper bound of 5.2 mya and a lower bound of 4.3 mya (corresponding approximately to

ʹͲʹ

the span of time from volcano emergence to shield completion for the oldest high-elevation

ʹͲ͵

Hawaiian Island, Kaua’i, as estimated by Obbard et al., 2012), and a mean of 4.7 mya. As

ʹͲͶ

secondary calibration points we used two dates estimated by Roalson and Roberts (2016) based

ʹͲͷ

on a combination of external fossils and geologic ages. The Cyrtandra crown was calibrated

ʹͲ͸

using a lognormal distribution, an upper bound of 21.3 mya and a lower bound of 4.7 mya

ʹͲ͹

(approximating the 95% highest posterior density of Roalson & Roberts (2016)), and a mean of

 

ͳͲ

ʹͲͺ

11.1 mya. The Pacific Cyrtandra crown was calibrated using a lognormal prior, an upper bound

ʹͲͻ

of 9.4 mya and a lower bound of 4.1 mya (approximating the 95% highest posterior density), and

ʹͳͲ

a mean of 6.4 mya. Convergence of runs and adequacy of MCMC sampling were checked using

ʹͳͳ

Tracer v. 1.6 (Rambaut et al., 2014), ensuring that ESS values were >200 for each parameter.

ʹͳʹ

After removing 25% of the trees as burn-in, a maximum clade-credibility tree was constructed

ʹͳ͵

using TreeAnnotator v. 2.3.2 (Drummond et al., 2012). The maximum clade credibility tree was

ʹͳͶ

then visualized in FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/), along with mean

ʹͳͷ

node heights, and the 95% highest posterior density interval of each node.

ʹͳ͸

2.5 Ancestral area estimation

ʹͳ͹

The package BioGeoBEARS (BioGeography with Bayesian (and likelihood)

ʹͳͺ

Evolutionary Analysis in R Scripts’; Matzke, 2013a) was used to estimate ancestral areas for

ʹͳͻ

Pacific Cyrtandra under the DEC (Dispersal-Extinction-Cladogenesis; Ree et al., 2005; Ree and

ʹʹͲ

Smith, 2008), DIVA (Dispersal-Vicariance Analysis; Ronquist, 1997) and BayArea (Bayesian

ʹʹͳ

Inference of Historical Biogeography for Discrete Areas; Landis et al., 2013) models in the R

ʹʹʹ

statistical environment (R Development Core Team, 2014). Each of the different models allows

ʹʹ͵

for a subset of biogeographic possibilities, enabling us to explore the role of different phenomena

ʹʹͶ

such as dispersal, vicariance, and extinction. These biogeographic processes are implemented in

ʹʹͷ

a maximum likelihood framework, and thus differ somewhat from the original parsimony based

ʹʹ͸

DIVA method and Bayesian BayArea method. We therefore follow Matzke (2013b) in referring

ʹʹ͹

to these models as DIVA-like and BayArea-like. We also explored the influence of two free

ʹʹͺ

parameters in BioGeoBEARS that are likely important for understanding the biogeographic

ʹʹͻ

history of island organisms: founder events (“j” parameter; Matzke, 2014), and dispersal distance

ʹ͵Ͳ

(“x” parameter; Van Dam and Matzke, 2016). Distances between areas were determined using

 

ͳͳ

ʹ͵ͳ

ArcGIS software (ESRI, 2011), with x being defined as the distance between the centers of two

ʹ͵ʹ

areas measured in kilometers. Distances were then rescaled (dividing by the smallest distance),

ʹ͵͵

such that the units of measurement would not influence the outcome of likelihood searches. The ultrametric BEAST tree was pruned to include only a single representative of each

ʹ͵Ͷ ʹ͵ͷ

species (except for those species that have multi-island distributions, for which we included one

ʹ͵͸

representative from each island). Each taxon was assigned to a set of 16 geographic regions

ʹ͵͹

based on collection localities: Southeast Asia (including Borneo, Malaysia, Sumatra, Java,

ʹ͵ͺ

Sulawesi, and the Philippines), Papua New Guinea, Bismarck Archipelago, Solomon Islands,

ʹ͵ͻ

Vanuatu, Australia, Loyalty Islands, Micronesia, Fiji, Samoa, Tonga, Wallis and Futuna, Society

ʹͶͲ

Islands, Austral Islands, Marquesas Islands, and the Hawaiian Islands. Outgroup taxa were

ʹͶͳ

removed so as to not influence the analysis. Although a few species are found in multiple regions

ʹͶʹ

(C. erectiloba, C. subulibractea, C. samoensis), we chose to treat these taxa as single-region

ʹͶ͵

populations due to the fact that these taxa are not monophyletic (see results). We set the

ʹͶͶ

maximum number of areas (i.e., the number of allowed geographic ranges that can be explored

ʹͶͷ

together in state space) to two, and then compared these models for statistical fit using the AIC

ʹͶ͸

and a Likelihood Ratio Test (LRT). Lastly, we used BioGeoBEARS to perform a biogeographic

ʹͶ͹

stochastic mapping (BSM) analysis (Matzke, 2016). We conducted 500 stochastic mapping

ʹͶͺ

simulations on the best model to get event counts for biogeographic processes involving

ʹͶͻ

anagenesis (range switching and range expansion dispersal) and cladogenesis (narrow sympatry,

ʹͷͲ

subset sympatry, widespread sympatry, vicariance, and founder events). In addition to estimating the biogeographic history of Cyrtandra across the Pacific, we

ʹͷͳ ʹͷʹ

performed a second BioGeoBEARS analysis to estimate ancestral areas and biogeographic

ʹͷ͵

events within a single archipelago. The main Hawaiian Islands were selected for this analysis for

 

ͳʹ

ʹͷͶ

two reasons: 1) the geologically young age of the main Hawaiian Islands means that signal of

ʹͷͷ

colonization history is less likely to have been erased by extinction, and 2) our taxon sampling

ʹͷ͸

for this archipelago was rigorous (38 of ca. 60 species). For this analysis we further pruned our

ʹͷ͹

dataset to include 47 accessions representing 39 taxa from the monophyletic Hawaiian group.

ʹͷͺ

Taxa were assigned to the main high-elevation Hawaiian Islands based on known collection

ʹͷͻ

localities: Kaua‘i, O‘ahu, Maui Nui (referring to the islands of Maui, Moloka‘i, and LƗna‘i,

ʹ͸Ͳ

which were connected as a single island for most of their history; Funk and Wagner, 1995), and

ʹ͸ͳ

Hawai‘i Island. Although a few species are found on multiple islands (C. hawaiensis, C.

ʹ͸ʹ

platyphylla, C. paludosa var. paludosa), we chose to treat these as single-island populations due

ʹ͸͵

to the fact that these taxa are not monophyletic (see results). The maximum number of areas was

ʹ͸Ͷ

set to four, and model selection and BSM analyses were carried out as above.

ʹ͸ͷ ʹ͸͸

3. Results

ʹ͸͹

3.1 Phylogenetic relationships In total, 603 new sequences were generated for this study and 162 were incorporated

ʹ͸ͺ ʹ͸ͻ

from GenBank. Our final data matrix consisted of 163 accessions representing 121 Cyrtandra

ʹ͹Ͳ

taxa, along with three species of Aeschynanthus and two species of Agalmyla as outgroups

ʹ͹ͳ

(Table 1). Summary statistics for each marker are presented in the Supplementary Information

ʹ͹ʹ

(SI Table S2). Independent ML analyses of the nrITS, nrETS, Cyrt1, psbA-trnH, and rpl32-trnL

ʹ͹͵

regions were largely congruent, with exceptions involving the placement of several taxa at the

ʹ͹Ͷ

tips of the tree (C. hornei, C. cf. kandavuensis, C. sp. nov. 1, C. ciliata, C. samoensis). For these

ʹ͹ͷ

taxa, conflict was observed either between nuclear regions (nrITS + nrETS and Cyrt1) or

ʹ͹͸

between nuclear and plastid regions (nrITS + nrETS and rpl32-trnL). These incongruences are

 

ͳ͵

ʹ͹͹

few and generally involve very closely related species such that they do not affect the

ʹ͹ͺ

overarching interpretation of relationships among major clades of species nor the inference of

ʹ͹ͻ

historical biogeography within the genus. Combined analysis is thus warranted as noted below;

ʹͺͲ

however, we suggest caution in the interpretation of species-level relationships involving these

ʹͺͳ

taxa. Analysis of the concatenated nuclear and cp regions resulted in far greater phylogenetic

ʹͺʹ ʹͺ͵

structure than achieved by analysis of individual genic regions. In consequence, the remainder of

ʹͺͶ

this paper will focus on results from the concatenated matrix, which included five gene regions

ʹͺͷ

and 3,625 aligned base positions. ML and BI trees were identical in topology, with the exception

ʹͺ͸

of the two outgroups. In the ML analysis, Agalmyla is sister to Cyrtandra (node 1; Figure 1) and

ʹͺ͹

Aeschynanthus is sister to Agalmyla + Cyrtandra. In contrast, the BI analysis reconstructed

ʹͺͺ

Aeschynanthus and Agalmyla as sister clades that are together sister to Cyrtandra (Fig. 2). In

ʹͺͻ

both analyses the majority of the backbone as well as major clades were well supported (• 80 BS

ʹͻͲ

and • 0.95 PP; Fig. 1). Species from Borneo, Sumatra, Java, Sulawesi, the Philippines, Malaysia,

ʹͻͳ

Papua New Guinea, Bismarck Archipelago, Solomon Islands, Vanuatu, and Australia are placed

ʹͻʹ

in several clades at the base of the tree (nodes 2–7; Fig. 1), and we follow Clark et al. (2013) in

ʹͻ͵

referring to this as the ‘Southeast Asian grade.’ The Southeast Asian grade includes the

ʹͻͶ

following: 1) a Borneo/Malaysia/Sumatra clade (node 14; 84 BS, 1.0 PP), 2) a Papua New

ʹͻͷ

Guinea/Solomon Islands/Vanuatu/Australia clade/Bismarck Archipelago clade (node 15; 100

ʹͻ͸

BS; 1.0 PP), 3) a Philippines/Solomon Islands clade (node 16; 87 BS, 1.0 PP), 4) a

ʹͻ͹

Sulawesi/Philippines/Java clade (node 17; 100 BS, 1.0 PP), 5) a single species from Papua New

ʹͻͺ

Guinea (node 6; 92 BS; 1.0 PP, and 6) a Solomon Islands clade (node 18; 100 BS, 1.0 PP). The Pacific clade comprises species from Fiji, the Hawaiian Islands, the Solomon Islands

ʹͻͻ

 

ͳͶ

͵ͲͲ

Samoa, the Marquesas Islands, Vanuatu, the Loyalty Islands, Wallis and Futuna, the Society

͵Ͳͳ

Islands, the Austral Islands, and Micronesia. The Fiji A clade contains a single species, C.

͵Ͳʹ

taviunensis, which is supported as sister to all remaining members of the Pacific clade (node 8;

͵Ͳ͵

99 BS, 1.0 PP). Three species were recovered as belonging to the Fiji B clade (node 19; 100 BS,

͵ͲͶ

1.0 PP), although the placement of this clade as sister to the Hawaiian group was only weakly

͵Ͳͷ

supported in the BI analysis (node 20; 29 BS, 0.56 PP). The Hawaiian group is supported as

͵Ͳ͸

monophyletic (node 21; 100 BS, 1.0 PP) but resolution within this clade is poor with few

͵Ͳ͹

species-level relationships being well supported in both ML and BI analyses. Lastly, a sister

͵Ͳͺ

relationship between the Fiji B/Hawaiian clade and the Solomon Islands E/South Pacific clade is

͵Ͳͻ

well supported in both ML and BI analyses (node 9; 92 BS, 1.0 PP). Four Samoan clades were supported: Samoa A is sister to the Marquesas group (node 22;

͵ͳͲ ͵ͳͳ

100 BS, 1.0 PP), whereas the Samoa B, C, and D clades are nested within a larger clade

͵ͳʹ

comprised of species from eight Pacific island regions (node 23; 100 BS, 1.0 PP). The Samoa B

͵ͳ͵

and C lineages consist of the paraphyletic C. samoensis, which is supported in two clades that are

͵ͳͶ

sister (94 BS, 1.0 PP). Accessions of C. samoensis from Samoa (Ofu island) and Tonga are

͵ͳͷ

placed sister to C. futunae from Wallis and Futuna (100 BS, 1.0 PP), whereas C. samoensis from

͵ͳ͸

Samoa (Upolu island) is sister to a single species from Vanuatu (99 BS, 1.0 PP), and these are in

͵ͳ͹

turn sister to two species from Micronesia (100 BS, 1.0 PP). The Samoa D lineage and Society

͵ͳͺ

Islands A clade form a strongly supported clade (100 BS, 1.0 PP). We found strong support for

͵ͳͻ

monophyly of the Marquesas group (87 BS, 1.0 PP), which comprises two supported clades

͵ʹͲ

(both 100 BS, 1.0 PP) and a possible third clade based on C. kenwoodii. Placement of the Solomon Islands E clade (node 10) and the Samoa A + Marquesas clade

͵ʹͳ ͵ʹʹ

(node 11) were not supported in the ML analysis (44 BS and 49 BS, respectively), and only

 

ͳͷ

͵ʹ͵

moderately supported in the BI analysis (0.90 PP and 0.91 PP, respectively). Seven additional

͵ʹͶ

Fijian clades were strongly supported (Fiji E–K; • 80 BS, • 0.95 PP), although relationships

͵ʹͷ

among these clades could not be resolved. Lastly, the Society Islands B clade and a single

͵ʹ͸

species from the Austral Islands are supported as together monophyletic (81 BS, 1.0 PP), and are

͵ʹ͹

placed sister to the Fiji H clade with weak support in the Bayesian analysis (46 BS, 0.73 PP).

͵ʹͺ

3.2 Divergence time estimates Divergence time estimates suggest that the genus Cyrtandra split from its closest

͵ʹͻ ͵͵Ͳ

relatives ~26 mya (18–35, 95% HPD) during the Oligocene. The crown age of Cyrtandra is

͵͵ͳ

estimated to be ~17 mya (13–22, 95% HPD), and the crown age of the Pacific clade is estimated

͵͵ʹ

to be ~9 mya (7–11, 95% HPD) (Fig. 2; Table 3). All major crown group lineages of Pacific

͵͵͵

Cyrtandra appear to have emerged within the last five million years (e.g., Fiji B = 3 mya,

͵͵Ͷ

Hawaiian Islands = 5 mya, Solomon Islands E = 3 mya, Samoa A = 3 mya, Marquesas Islands =

͵͵ͷ

3 mya, Society Islands B = 2 mya).

͵͵͸

3.3 Ancestral area estimation In the BioGeoBEARS analysis of Pacific Cyrtandra, significant improvement in the

͵͵͹ ͵͵ͺ

likelihood of the three standard models (DEC, DIVA-like, and BayArea-like) was seen when

͵͵ͻ

founder events and distance parameters were added (SI Table S3). Of the 12 models evaluated,

͵ͶͲ

three were plausible (i.e., ǻAIC values were < 7; Burnham et al., 2011): DEC+j+x, DIVA-

͵Ͷͳ

like+j+x, and BayArea-like+j+x. Given that all three of these models were broadly congruent

͵Ͷʹ

and varied only slightly in the probabilities estimated for the backbone nodes comprising the

͵Ͷ͵

Southeast Asian grade, for simplicity we present only the results from the second best model

͵ͶͶ

(BayArea-like+j+x) based on the more conservative estimation of these backbone nodes

͵Ͷͷ

(Southeast Asia/New Guinea vs. Southeast Asia or New Guinea). The BayArea-like+j+x model

 

ͳ͸

͵Ͷ͸

was significantly better than the null model (BayArea-like) according to a LRT (LnL = −130.98

͵Ͷ͹

vs. LnL = −179.00, df = 2, p < 0.001). The resulting parameters of the BayArea-like+j+x model

͵Ͷͺ

include: anagenetic dispersal rate (d) = 0, extinction rate (e) = 0.1315, cladogenetic dispersal rate

͵Ͷͻ

(j) = 0.0565, and dispersal distance (x) = −1.4099. Given the parameters of the BayArea-like+j+x

͵ͷͲ

model, results from the biogeographic stochastic mapping in BioGeoBEARS suggest that 100%

͵ͷͳ

of all cladogenetic events between island archipelagos involved founder events, with a mean of

͵ͷʹ

24.75 ± 0.84 founder events identified in the present phylogeny (Fig. 3; Table 4).

͵ͷ͵

Ancestral area estimation under the BayArea-like+j+x model indicates that Cyrtandra

͵ͷͶ

most likely originated in Southeast Asia/New Guinea (Fig. 3). Two founder events from New

͵ͷͷ

Guinea resulted in the Bismarck Archipelago clade and the Solomon Islands A clade. Two

͵ͷ͸

founder events from the Bismarck Archipelago gave rise to the Solomon Islands B clade and the

͵ͷ͹

only known Australian species, although this result is tentative (47% probability). One founder

͵ͷͺ

event from the Solomon Islands gave rise to the Vanuatu A clade, while a founder event from

͵ͷͻ

Australia resulted in the Vanuatu B clade. The Solomon Islands C and D clades were estimated

͵͸Ͳ

to be the result of two founder events from a combined Southeast Asia/New Guinea region. A

͵͸ͳ

second founder event from the Solomon Islands resulted in the Fijian species. The ancestral area

͵͸ʹ

for the Pacific clade is estimated to be Fiji with a 98% probability. Subsequently, there were six

͵͸͵

founder events from Fiji to other regions in the Pacific: one to the Solomon Islands (Solomon

͵͸Ͷ

Islands E), two to Samoa (Samoa A and Samoa B/C/D), one to the Society Islands (Society

͵͸ͷ

Islands B), one to the Loyalty Islands, and one to the Hawaiian Islands. The Loyalty

͵͸͸

Islands/Vanuatu C clade could be the result of a founder event from either the Loyalty Islands or

͵͸͹

Vanuatu (52% vs. 48% probability). Dispersal from Samoa also resulted in a total of six founder

͵͸ͺ

events: one to Vanuatu (Vanuatu D), one to Tonga, one to Wallis and Futuna, one to Micronesia,

 

ͳ͹

͵͸ͻ

one to the Society Islands (Society Islands A), and one to the Marquesas Islands. One founder

͵͹Ͳ

event from the Society Islands to the nearby Austral Islands resulted in a single species. Phylogenetic relationships within the monophyletic Hawaiian Cyrtandra were largely

͵͹ͳ ͵͹ʹ

unresolved in our ML and BI analyses, such that results from our BioGeoBEARS analyses for

͵͹͵

this group must be interpreted as preliminary until greater resolution can be achieved for this

͵͹Ͷ

young lineage. Similar to the analysis performed for Pacific Cyrtandra, there was significant

͵͹ͷ

improvement in the likelihood of the three standard models with the addition of founder events

͵͹͸

and dispersal distance parameters in Hawaiian Cyrtandra (SI Table S4). All of the models

͵͹͹

evaluated except for DEC, DIVA-like, and BayArea-like can be considered plausible (i.e., ǻAIC

͵͹ͺ

values were < 7; Burnham et al., 2011). All plausible models exhibited only very minor

͵͹ͻ

differences in ancestral area probabilities, particularly for the two basal-most nodes that were

͵ͺͲ

largely unresolved in all models (i.e., probabilities were ” 32% for all areas). Here we present the

͵ͺͳ

results from the second-best model (BayArea-like+j+x) as the estimated ancestral areas for these

͵ͺʹ

basal nodes seem more conservative than those estimated in the other plausible models

͵ͺ͵

(Kaua‘i/O‘ahu vs. Kaua‘i/O‘ahu/Maui Nui, O‘ahu, or Kaua‘i). The BayArea-like+j+x model was

͵ͺͶ

significantly better than the null model (BayArea) according to a LRT (LnL = −35.91 vs. LnL =

͵ͺͷ

−44.87, df = 2, p < 0.001). The resulting parameters of the BayArea-like+j+x model include:

͵ͺ͸

anagenetic dispersal rate (d) = 0, extinction rate (e) = 1.1121, cladogenetic dispersal rate (j) =

͵ͺ͹

0.1041, and dispersal distance (x) = −1.2988 (Fig. 4; SI Table S4). Given the parameters of the

͵ͺͺ

BayArea-like+j+x model, results from the biogeographic stochastic mapping in BioGeoBEARS

͵ͺͻ

suggest that the ancestral area for Cyrtandra in Hawaii is largely unresolved, with only a 23%

͵ͻͲ

probability of a Kaua‘i/O‘ahu origin (Fig. 4). A mean of 10.18 ± 1.01 founder events were

͵ͻͳ

recovered, corresponding to the following: Kaua‘i to Maui Nui (1), Maui Nui to Kaua‘i (1),

 

ͳͺ

͵ͻʹ

O‘ahu to Maui Nui (2), Maui Nui to O‘ahu (1), Maui Nui to Hawai‘i (3), and Hawai‘i to Maui

͵ͻ͵

Nui (2; Fig. 4; Table 4).

͵ͻͶ ͵ͻͷ

4. Discussion

͵ͻ͸

4.1 Phylogenetic relationships within Pacific Cyrtandra The maximum likelihood and Bayesian analyses for the concatenated five-gene dataset

͵ͻ͹ ͵ͻͺ

largely corroborated the phylogenetic relationships revealed in previous studies (Clark et al.,

͵ͻͻ

2008, 2009, 2013) and also provided resolution for some previously unresolved aspects.

ͶͲͲ

Specifically, our analyses: 1) resolved key nodes in the backbone of the tree, 2) recovered a sister

ͶͲͳ

relationship between the Fiji A clade and the remaining members of the Pacific clade, 3) clarified

ͶͲʹ

the placement of taxa from previously un-sampled regions including Papua New Guinea, the

ͶͲ͵

Bismarck Archipelago, Vanuatu, the Loyalty Islands, Wallis and Futuna, and the Austral Islands,

ͶͲͶ

and 4) recovered 11 Fijian clades. Uncertainty remains regarding the closest relative to Cyrtandra as the ML and BI

ͶͲͷ ͶͲ͸

analyses were incongruent. Roalson and Roberts (2016) included data for 26 gene regions and

ͶͲ͹

numerous genera from across Gesneriaceae and were unable to resolve the sister to Cyrtandra

ͶͲͺ

with confidence; Agalmyla was sister to Cyrtandra but support was weak (aLRT = 70) or lacking

ͶͲͻ

(BS = 8). Additional sampling of taxa and genic regions is clearly needed to resolve this

ͶͳͲ

relationship. In the present study, there was also evidence for topological incongruences between

Ͷͳͳ

individual gene trees, mainly involving several taxa at the tips of the phylogeny. These

Ͷͳʹ

incongruences may be due to incomplete lineage sorting, hybridization and/or introgression

Ͷͳ͵

among taxa that have diverged only very recently (i.e., < 5 mya). Pillon et al. (2013b) also found

ͶͳͶ

evidence for incongruence among gene trees in Hawaiian Cyrtandra, and cited deep coalescence,

 

ͳͻ

Ͷͳͷ

introgression, and hybridization as possible causes. Molecular (Smith et al., 1996) and

Ͷͳ͸

morphological (Johnson et al., 2015) evidence for hybridization exists for several species pairs in

Ͷͳ͹

the young Hawaiian lineage. Additional evidence for these issues may include the lack of

Ͷͳͺ

monophyly for most multi-island taxa in the present study (C. erectiloba, C. subulibractea, C.

Ͷͳͻ

leucantha, C. richii, C. pogonantha, C. samoensis, C. hawaiensis, C. platyphylla, C. paludosa

ͶʹͲ

var. paludosa). Cautious interpretation of phylogenetic relationships, particularly among those of

Ͷʹͳ

recently diverged species, is therefore warranted. Species comprising the five clades of Cyrtandra from the Solomon Islands show far

Ͷʹʹ Ͷʹ͵

greater morphological variation than clades endemic to the more remote Pacific archipelagos.

ͶʹͶ

The Solomon Islands are in close proximity to several floristically diverse source areas including

Ͷʹͷ

Papua New Guinea, Australia, and the Southwest Pacific Islands (Thorne, 1969; van Balgooy,

Ͷʹ͸

1971; Mueller-Dombois and Fosberg, 1998). Clark et al. (2013) recognized the Solomon Islands

Ͷʹ͹

as a zone of intermediacy between Southeast Asia and the Pacific due to the range of

Ͷʹͺ

morphological characters exhibited by taxa there, as well as the phylogenetic placement of

Ͷʹͻ

species from the Solomon Islands as either part of the Southeast Asian grade or nested within the

Ͷ͵Ͳ

Pacific clade. The present study finds the same phylogenetic pattern in species from Vanuatu,

Ͷ͵ͳ

with two species being placed within the Southeast Asian grade, and two species nested within

Ͷ͵ʹ

the Pacific clade. While the floristic affinities of Vanuatu are more strongly South Pacific, there

Ͷ͵͵

is greater influence from New Guinea than found, for example, in Fiji (Mueller-Dombois and

Ͷ͵Ͷ

Fosberg, 1998). We suggest that Vanuatu may also be considered a region of intermediacy

Ͷ͵ͷ

between Southeast Asia and the Pacific. Perhaps the most important phylogenetic result of this study was the strong support for C.

Ͷ͵͸ Ͷ͵͹

taviunensis from Fiji as sister to the remaining members of the Pacific clade. This result is

 

ʹͲ

Ͷ͵ͺ

intriguing given that C. taviunensis is one of only seven species of Cyrtandra known to have

Ͷ͵ͻ

orange fleshy fruits, all of which are endemic to Fiji and Samoa. In Gillett’s (1967) treatment of

ͶͶͲ

Fijian Cyrtandra it was hypothesized that C. taviunensis (endemic to Taveuni, Fiji) was sister to

ͶͶͳ

C. montana (not sampled here; endemic to Viti Levu, Fiji). This was based on a number of

ͶͶʹ

shared traits such as foliage indument, flower structure, and orange fruits. Examination of

ͶͶ͵

herbarium material suggests that these species do indeed share considerable morphological

ͶͶͶ

affinities and are likely each other’s closest relatives. The only other known orange-fruited Fijian

ͶͶͷ

species, C. cephalophora (Fiji F clade), is very different morphologically from C. taviunensis

ͶͶ͸

and C. montana, and was hypothesized by Gillett (1967) to represent a separate dispersal event

ͶͶ͹

from mainland regions. However, the present study does not support a separate introduction of

ͶͶͺ

C. cephalophora as predicted. Rather, C. cephalophora is nested within a clade comprised of

ͶͶͻ

white-fruited Fijian species. Given that the two orange-fruited Fijian species sampled in the

ͶͷͲ

present study are strongly supported as belonging to separate clades, two independent origins of

Ͷͷͳ

orange fruits in Fijian Cyrtandra are inferred. The Samoa A clade comprises all other orange-

Ͷͷʹ

fruited species that have been sampled to date. However, at least one other species with orange

Ͷͷ͵

fruits is known in Samoa (C. angustivenosa from Savai‘i), and fruits of several other species are

ͶͷͶ

undocumented. Thus, it remains unknown whether orange fruits evolved once or multiple times

Ͷͷͷ

among the Samoan Cyrtandra. Our results confirm the existence of the Fiji B clade, as proposed by Clark et al. (2008,

Ͷͷ͸ Ͷͷ͹

2009, 2013) and expand the clade by two additional species. The Fiji B clade is of interest in that

Ͷͷͺ

it may be closely related to the monophyletic Hawaiian lineage, although we did not find strong

Ͷͷͻ

support for a sister relationship. This node has been particularly difficult to resolve (see Cronk et

Ͷ͸Ͳ

al., 2005; Clark et al., 2008, 2009, 2013), and is possibly reflective of a recent and rapid radiation

 

ʹͳ

Ͷ͸ͳ

from the South Pacific to the Hawaiian Islands. Monophyly of the Hawaiian lineage suggests a

Ͷ͸ʹ

single introduction, a pattern that is common among many of the large plant radiations in the

Ͷ͸͵

Hawaiian Islands (reviewed in Keeley and Funk, 2011), although several genera have colonized

Ͷ͸Ͷ

the archipelago multiple times (Scaevola, Howarth et al., 2003; Santalum, Harbaugh and

Ͷ͸ͷ

Baldwin, 2007; Coprosma, Cantley et al., 2014). The Hawaiian clade of Cyrtandra was poorly

Ͷ͸͸

resolved overall, perhaps because it is an evolutionarily young lineage. However, there does

Ͷ͸͹

appear to be a trend for species to group by island, as was also recognized by Clark et al. (2009). In contrast to Clark et al. (2013) we found only moderate to no support (BI and ML

Ͷ͸ͺ Ͷ͸ͻ

analyses, respectively) for the placement of the Solomon Islands E clade and the Samoa

Ͷ͹Ͳ

A/Marquesas Islands clade. The low support for the placement of these nodes is possibly a result

Ͷ͹ͳ

of increased sampling and genic data across a relatively large but genetically similar genus. Our

Ͷ͹ʹ

results support that the sampled Marquesas Islands species are together monophyletic and

Ͷ͹͵

comprise at least two distinct clades, with a possible third clade based on the unsupported

Ͷ͹Ͷ

placement of C. kenwoodii. Species in the Marquesas seem to group based on morphological

Ͷ͹ͷ

similarities in the calyx (Wagner et al., 2013) as well as island proximity, with species from the

Ͷ͹͸

older islands of the Northern Marquesas forming a clade (C. jonesii and C. thibaultii) and species

Ͷ͹͹

from the younger islands of the Southern Marquesas forming a clade (C. feaniana, C. ootensis,

Ͷ͹ͺ

and C. tahuatensis). Two other species from the Marquesas Islands that were not included in the

Ͷ͹ͻ

present study (C. toviana, C. revoluta) are highly distinct morphologically from other Marquesas

ͶͺͲ

Islands species (Wagner et al., 2013), such that the possibility of more than one introduction of

Ͷͺͳ

Cyrtandra to the archipelago still exists. The isolated location of the Marquesas Islands has

Ͷͺʹ

resulted in only a single introduction of many Pacific plant genera to the archipelago (Scaevola,

Ͷͺ͵

Howarth et al., 2003; Astelia, Birch and Keeley, 2013; Coprosma, Cantley et al., 2016), with the

 

ʹʹ

ͶͺͶ

exception of two independent introductions of Melicope from the Hawaiian Islands (Appelhans

Ͷͺͷ

et al., 2014). Lastly, while only three of the 16 species endemic to the Society Islands were

Ͷͺ͸

sampled, these species formed two distinct clades suggesting that increased sampling in this

Ͷͺ͹

region may reveal additional clades. Polyphyly of congeneric taxa has been shown to be

Ͷͺͺ

common in the Society Islands, suggesting multiple independent colonizations of the archipelago

Ͷͺͻ

by many plant genera (Hembry and Balukjian, 2016).

ͶͻͲ

A clade that is particularly complex in our analysis (node 23; Fig. 1) comprises species

Ͷͻͳ

from eight Pacific regions: Vanuatu, Fiji, Samoa, Tonga, Society Islands, Micronesia, Loyalty

Ͷͻʹ

Islands, and Wallis and Futuna. Gillett (1973) first recognized this group (which he termed the

Ͷͻ͵

“Cyrtandra cymosa group”) as a species complex with exceedingly high dispersability, and

ͶͻͶ

surmised that its existence challenged the idea that Cyrtandra species are always narrow

Ͷͻͷ

endemics that rarely extend their range beyond a single island or archipelago. This complex, as

Ͷͻ͸

proposed by Gillett (1973), consisted of nine morphologically similar species: C. cymosa

Ͷͻ͹

(Vanuatu), C. cominsii (Vanuatu), C. urvillei (Micronesia), C. mareensis (Loyalty Islands), C.

Ͷͻͺ

rotumaensis (Rotuma), C. futunae (Wallis and Futuna), C. tempestii (Fiji), C. samoensis (Samoa,

Ͷͻͻ

Niue, and Tonga), and C. raratongensis (Cook Islands). Clark et al.’s (2009) phylogeny

ͷͲͲ

confirmed that C. samoensis (Samoa, Tonga), C. urvillei (Micronesia), and C. kusaimontana

ͷͲͳ

(Micronesia) formed a strongly supported clade, which the authors termed the “Cyrtandra

ͷͲʹ

samoensis complex.” Cyrtandra samoensis was identified as being paraphyletic in previous

ͷͲ͵

studies (Clark et al., 2008, 2009, 2013) and this result is also supported in the present study.

ͷͲͶ

Our phylogenetic results include additional taxa in this clade that do not particularly

ͷͲͷ

share similar characteristics. We propose that the pattern of species relationships within this

ͷͲ͸

clade may be explained by variation in dispersability, which is linked to habitat preference. Of

 

ʹ͵

ͷͲ͹

the 121 Cyrtandra taxa sampled in the present study, only seven species are known to occur at or

ͷͲͺ

near sea level. Of these seven species (C. fulvovillosa, C. samoensis, C. futunae, C. sp. ‘hiu’, C.

ͷͲͻ

sp. nov. 2, C. sp. nov. 3, C. mareensis), five belong to the clade described here. Species growing

ͷͳͲ

near the coast may be dispersed between distant islands by migratory shore birds, which

ͷͳͳ

regularly fly over vast ocean expanses. Following colonization, coastal populations may

ͷͳʹ

diversify and adapt to upland forest habitats (see Jønsson et al., 2014). Species growing in upland

ͷͳ͵

rainforests may in turn be dispersed by forest birds, which typically have smaller range sizes and

ͷͳͶ

are less likely to fly between islands (Gillespie et al., 2012). Under this scenario, the pattern of

ͷͳͷ

species relationships in this clade can be explained by invoking island colonization by coastal

ͷͳ͸

species with high rates of inter-island dispersal, which later diversified and gave rise to upland

ͷͳ͹

rainforest species with low rates of inter-island dispersal.

ͷͳͺ

The vast majority of Pacific Cyrtandra species are endemic to upland rainforests but it is

ͷͳͻ

possible that coastal species were more common in the past. Changes in land use practices have

ͷʹͲ

led to the destruction or alteration of native coastal and lowland forest habitat across the Pacific,

ͷʹͳ

resulting in increasing numbers of endangered and extinct taxa. According to the IUCN (2016),

ͷʹʹ

two of the coastal species discussed here are listed as critically endangered (C. raratongensis, C.

ͷʹ͵

mareensis); two others that are in the process of being described (C. sp. nov. 2, C. sp. nov. 3)

ͷʹͶ

also qualify as critically endangered due to their limited geographic range and small population

ͷʹͷ

sizes (M. Johnson, pers. obs.).

ͷʹ͸

4.2 Divergence times Recent age estimates suggest that Gesneriaceae emerged ca. 70 mya in the Andean region

ͷʹ͹ ͷʹͺ

of South America, followed by dispersal to eastern Asia/Southeast Asia where the

ͷʹͻ

Didymocarpoideae lineage (2100+ species, including Cyrtandra) evolved ca. 67 mya (Roalson

 

ʹͶ

ͷ͵Ͳ

and Roberts, 2016). Cyrtandra is estimated to be one of the more recently derived genera within

ͷ͵ͳ

the Didymocarpoideae (Roalson and Roberts, 2016), which is in line with our estimate that the

ͷ͵ʹ

crown group of Cyrtandra emerged ca. 17 mya. Notably, our divergence time estimates are

ͷ͵͵

younger than those previously estimated using the likelihood-based program r8s (Clark et al.,

ͷ͵Ͷ

2009). However, our mean divergence time estimates for Cyrtandra are within the 95% HPD for

ͷ͵ͷ

dates reported by Roalson and Roberts (2016; Table 3) using BEAST. Increased taxon sampling

ͷ͵͸

in the present study may account for the observed differences in age estimates. The fact that all major crown group lineages of Pacific Cyrtandra emerged only within

ͷ͵͹ ͷ͵ͺ

the last five million years suggests an accelerated rate of speciation at the end of the

ͷ͵ͻ

Miocene/beginning of the Pliocene, although we did not test this in the present study. The rapid

ͷͶͲ

radiation of Cyrtandra in the Pacific is likely related to the availability of habitat (see Yoder et

ͷͶͳ

al., 2010) that coincided with the emergence of many Pacific islands within the last five million

ͷͶʹ

years, including the Marquesas Islands (5.5–0.4 mya), Samoa (5.2–1.0 mya), the main Hawaiian

ͷͶ͵

Islands (5.0–0.3 mya), the Society Islands (4.5–0.6 mya), and the Fijian islands of Kadavu (3.4

ͷͶͶ

mya) and Taveuni (3.0–0.7 mya) (island ages reviewed in Neall and Trewick, 2008). Changes in

ͷͶͷ

climate and associated range shifts may have also facilitated within-island divergence on older

ͷͶ͸

islands such as Viti Levu (29–24 mya; Taylor et al., 2000) in Fiji. Our divergence time estimates suggest that Cyrtandra colonized the Hawaiian Islands 4.6

ͷͶ͹ ͷͶͺ

mya, shortly after the emergence of the oldest high-elevation island (Kaua‘i: 5.02 mya; Obbard

ͷͶͻ

et al., 2012). Island formation can be used to infer the maximum age of clades that are endemic

ͷͷͲ

to those islands, but we acknowledge that our use of the main Hawaiian Islands as a calibration

ͷͷͳ

point may have resulted in the overestimation of the age of some lineages (reviewed in Heads,

ͷͷʹ

2011). Although it may be assumed that colonization can take place soon after island emergence

 

ʹͷ

ͷͷ͵

for some plant taxa (e.g., Metrosideros polymorpha var. incana is ecologically adapted to

ͷͷͶ

colonize young lava flows in Hawaii; Morrison and Stacy, 2014), species of Cyrtandra would

ͷͷͷ

likely require a forest overstory to exist prior to successful colonization and establishment. As a

ͷͷ͸

closed canopy forest could potentially take thousands of years to develop, taxa endemic to a

ͷͷ͹

given island would have to be at least several thousand years younger than the island itself.

ͷͷͺ

Additionally, if the island is subject to ongoing volcanic activity that regularly extirpates existing

ͷͷͻ

forest communities, taxa could potentially be much younger than the islands that they are

ͷ͸Ͳ

endemic to (see Obbard et al., 2012). As such, the delay between island emergence and

ͷ͸ͳ

permanent colonization may be substantial. Lastly, a number of studies have found island taxa to

ͷ͸ʹ

be older than the islands they are endemic to (Jønsson et al., 2010; Le Péchon et al., 2015;

ͷ͸͵

Chomicki and Renner, 2016), a scenario that could be explained by taxa becoming extinct on

ͷ͸Ͷ

older islands after they colonized younger islands in the archipelago (reviewed in Heads, 2011).

ͷ͸ͷ

In the present study we do not find evidence of any such occurrence, but we acknowledge that

ͷ͸͸

these caveats exist and advise caution in the interpretation of age estimates presented for these

ͷ͸͹

island taxa.

ͷ͸ͺ

4.3 Historical biogeography Previous studies examining historical biogeography in Cyrtandra compared four methods

ͷ͸ͻ ͷ͹Ͳ

for inferring ancestral ranges: Fitch Parsimony, Stochastic Mapping (likelihood-based), DIVA,

ͷ͹ͳ

and DEC (Clark et al., 2008, 2009). These studies found DEC to be the best model based on its

ͷ͹ʹ

ability to incorporate information on island ages and prior hypotheses of range size and dispersal

ͷ͹͵

rate. A limitation at the time of these studies, the DEC model only included two parameters

ͷ͹Ͷ

(dispersal and extinction), and could not account for parameters such as founder events, which

ͷ͹ͷ

has more recently been shown to be relevant for estimating ancestral ranges in oceanic island

 

ʹ͸

ͷ͹͸

systems (Matzke, 2014). In addition, with just a single base model being used, it was not possible

ͷ͹͹

in previous studies to incorporate statistical tools for comparing and selecting models, such as

ͷ͹ͺ

the AIC and LRT. With recent advances in biogeographic inference methods, we have been able to revisit

ͷ͹ͻ ͷͺͲ

the estimation of ancestral ranges in Cyrtandra by comparing the DEC, DIVA-like, and

ͷͺͳ

BayArea-like models as implemented in BioGeoBEARS, and have added founder events and

ͷͺʹ

dispersal distance as free parameters to each base model. Results from our model comparisons

ͷͺ͵

using the AIC and LRT suggest that incorporating founder events and dispersal distance indeed

ͷͺͶ

improved model fit. The best model suggested that extinction, founder events, and dispersal

ͷͺͷ

distance have all played major roles in shaping species distribution patterns among Pacific

ͷͺ͸

archipelagos. The inferred ancestral area for Cyrtandra under the BayArea-like+j+x model is a

ͷͺ͹

combined Southeast Asia/New Guinea region. The inclusion of additional species from major

ͷͺͺ

regions within Southeast Asia (Atkins et al., in prep) will be necessary to determine the precise

ͷͺͻ

area of origin for Cyrtandra. Additional sampling in species-rich areas such as New Guinea may

ͷͻͲ

also result in revised ancestral area estimates for taxa such as C. baileyi from Australia, which is

ͷͻͳ

currently estimated as the result of a founder event from the Bismarck Archipelago but is

ͷͻʹ

geographically much closer to Papua New Guinea. The area of origin for the Pacific clade is estimated as Fiji with high probability (98%).

ͷͻ͵ ͷͻͶ

Our results concur with Clark et al.’s (2008, 2009) finding that Fiji and, to a lesser extent, Samoa

ͷͻͷ

is a possible biogeographic center of dispersal between Southeast Asia and the Pacific. Results

ͷͻ͸

from the BSM analysis in BioGeoBEARS found a total of six cladogenetic dispersal events each

ͷͻ͹

from Fiji and Samoa, resulting in remote island archipelagos across the Pacific being colonized

ͷͻͺ

by Cyrtandra. All cladogenesis among Pacific archipelagos was the result of founder events,

 

ʹ͹

ͷͻͻ

with dispersal being more frequent between archipelagos that are separated by shorter distances.

͸ͲͲ

However, the range of distances between island archipelagos that were involved in founder

͸Ͳͳ

events (575 km–5075 km) were all well above the threshold generally considered to constitute

͸Ͳʹ

long-distance dispersal between archipelagos (>200 km: Keppel et al., 2009; >100 km: Gillespie

͸Ͳ͵

et al., 2012). One of the most striking patterns to emerge from our biogeographic analysis of Pacific

͸ͲͶ ͸Ͳͷ

Cyrtandra was the strong asymmetry in the direction of founder events, with the majority of

͸Ͳ͸

dispersals occurring from a west to east direction. Only seven founder events were from east to

͸Ͳ͹

west, and a single founder event was from south to north. These dispersal patterns are

͸Ͳͺ

representative of the direction and frequency with which birds fly between islands in the South

͸Ͳͻ

Pacific (see Gillespie et al., 2012). Fruit doves (Ptilinopus) and imperial pigeons (Ducula) are

͸ͳͲ

forest-dwelling frugivorous birds that have high species diversity and a broad geographic

͸ͳͳ

distribution centered on New Guinea and the Pacific (Cibois et al., 2014, 2017). Given that the

͸ͳʹ

distribution of these birds mirrors that of Cyrtandra, dispersal by these genera seems a likely

͸ͳ͵

scenario. However, these genera are absent from the Hawaiian archipelago, such that dispersal to

͸ͳͶ

this isolated region may have involved migratory birds (e.g., the Pacific golden plover, Pluvialis

͸ͳͷ

fulva) traveling along the Central Pacific flyway (Gillespie et al., 2012). Inter-island dispersal of

͸ͳ͸

plants with fleshy fruits may also involve floatation across the open ocean, but the density of

͸ͳ͹

Pacific Cyrtandra fruits makes this mode of inter-island dispersal seem highly unlikely.

͸ͳͺ

However, Pacific Cyrtandra species are often found growing along steep ravines and waterways

͸ͳͻ

(M. Johnson, pers. obs.) and may be locally dispersed when fruits are washed downstream.

͸ʹͲ

Evidence for repeated dispersals, wherein the same geographic region was colonized

͸ʹͳ

multiple times by independent lineages, was also a prominent finding from the BSM results.

 

ʹͺ

͸ʹʹ

While this pattern has been shown in a number of other Pacific plant groups (Scaevola, Howarth

͸ʹ͵

et al., 2003; Melicope, Appelhans et al., 2014; Santalum, Harbaugh et al., 2007; Metrosideros,

͸ʹͶ

Papadopulos et al., 2011), only in Coprosma (8 or 9 geographic regions with repeated dispersals;

͸ʹͷ

Cantley et al., 2016) have repeated dispersals been more frequent than in Cyrtandra. Repeated

͸ʹ͸

dispersals of Cyrtandra were found for five geographic regions, with Papua New Guinea being

͸ʹ͹

colonized at least twice (Southeast Asia = 1 and the Solomon Islands = 1), the Solomon Islands

͸ʹͺ

five times (Papua New Guinea = 3, Bismarck Archipelago = 1, and Fiji = 1), Vanuatu four times

͸ʹͻ

(Solomon Islands = 1, Australia = 1, Samoa = 1, and the Loyalty Islands = 1), Samoa twice (Fiji

͸͵Ͳ

= 2), and the Society Islands twice (Fiji = 1 and Samoa = 1). The pattern of repeated dispersal

͸͵ͳ

presented here suggests that the chances of an island being colonized multiple times decreases as

͸͵ʹ

the distance from continental source regions increases. Our analysis of historical biogeography in Hawaiian Cyrtandra revealed that extinction,

͸͵͵ ͸͵Ͷ

founder events, and dispersal distance are all important in shaping the observed species

͸͵ͷ

distribution patterns within a single archipelago. Results from the BayArea-like+j+x model

͸͵͸

suggest that Cyrtandra initially colonized the geologically older high-elevation Hawaiian Islands

͸͵͹

(Kaua‘i: 5.02–4.24 mya, O‘ahu: 4.32–3.54 mya; Obbard et al., 2012). However, it is difficult to

͸͵ͺ

infer which of these two islands may have hosted the initial colonization event; Cyrtandra taxa

͸͵ͻ

endemic to Kaua‘i have the longest branches among the Hawaiian species, but the island of

͸ͶͲ

O‘ahu has the highest number of endemic Cyrtandra taxa. Dispersal events between the islands

͸Ͷͳ

of Kaua‘i and O‘ahu were not inferred in the BSM analysis, likely due to the uncertainty in

͸Ͷʹ

ancestral area estimations for the basal-most nodes in the Hawaiian clade. In the Hawaiian

͸Ͷ͵

Islands, the general pattern of stepping-stone dispersal from older to younger islands followed by

͸ͶͶ

geographic isolation and species divergence has been well documented in plants (Wagner and

 

ʹͻ

͸Ͷͷ

Funk, 1995; Fleischer et al., 1998; Percy et al., 2008; Pillon et al., 2013a). Younger islands may

͸Ͷ͸

offer better chances for successful colonization given the unoccupied (and sometimes novel)

͸Ͷ͹

habitat that is available on recently emerged volcanic substrates (Yoder et al., 2010; Shaw and

͸Ͷͺ

Gillespie, 2016). However, results from our BSM analysis suggest that dispersal from older to

͸Ͷͻ

younger islands is only slightly more frequent than is dispersal from younger to older islands (6

͸ͷͲ

vs. 4 dispersal events; Fig. 3). Dispersal from younger to older islands may reflect the ability of

͸ͷͳ

Cyrtandra species to colonize islands that have established ecosystems in place (see Clark et al.,

͸ͷʹ

2009). Lastly, in accordance with results from our Pacific Cyrtandra BSM analysis, we find that

͸ͷ͵

founder events account for all of the cladogenesis within the Hawaiian archipelago (Table 4; Fig.

͸ͷͶ

4). The distribution of Pacific Cyrtandra is somewhat similar to that of other well-studied

͸ͷͷ ͸ͷ͸

Pacific plant genera that are bird-dispersed, such as Coprosma (Cantley et al., 2016), Psychotria

͸ͷ͹

(Barrabé et al., 2014), Pittosporum (Gemmill et al., 2002), and Astelia (Birch and Keeley, 2013).

͸ͷͺ

However, several key differences can be seen among these highly dispersive groups. First, while

͸ͷͻ

Cyrtandra evolved in Southeast Asia and then colonized the Pacific via stepping-stone dispersal

͸͸Ͳ

from the east, other genera emerged in New Zealand, Australia and New Caledonia and then

͸͸ͳ

dispersed in a NE and NW direction into the Pacific. Second, while these genera successfully

͸͸ʹ

colonized the Fiji-Samoa region, this did not lead to subsequent colonization of more isolated

͸͸͵

Pacific archipelagos as in Cyrtandra (with the exception of Pittosporum which tentatively shows

͸͸Ͷ

a sister relationship between the Hawaiian and Fijian/Tongan species; Gemmill et al., 2002).

͸͸ͷ

Rather, these isolated regions were reached via other oceanic islands (e.g., the Austral Islands) or

͸͸͸

continental islands (e.g., New Zealand) through either direct dispersal or stepping-stone

͸͸͹

dispersal. Lastly, other Pacific plant genera are well represented in New Zealand, New Caledonia

 

͵Ͳ

͸͸ͺ

and Lord Howe Island while Cyrtandra is absent from these regions. The absence of Cyrtandra

͸͸ͻ

from these continental islands may be due to failure to disperse to these regions, although it

͸͹Ͳ

seems more likely that taxa failed to successfully establish and diversify. One potential

͸͹ͳ

explanation may be that Cyrtandra dispersed to these regions, but experienced strong ecological

͸͹ʹ

niche competition from closely related taxa (e.g., Coronanthera spp.), which colonized these

͸͹͵

continental islands and diversified within the forest understories prior to the evolution of the

͸͹Ͷ

Pacific clade of Cyrtandra (Woo et al., 2011). In contrast, Cyrtandra is the only genus within the

͸͹ͷ

Gesneriaceae family that occurs on oceanic islands west of the Solomon Islands (Burtt, 2001),

͸͹͸

and therefore would not have experienced competition pressure from closely related species with

͸͹͹

similar niche preferences on Pacific islands. Alternatively, New Zealand, New Caledonia and

͸͹ͺ

Lord Howe Island may be too arid for successful establishment of Cyrtandra, which is almost

͸͹ͻ

exclusively restricted to rainforests. The Pacific clade of Psychotria exhibits a similar

͸ͺͲ

distribution pattern in that there is a large number of shrub/epiphytic species found in rainforest

͸ͺͳ

understories throughout the Pacific (ca. 340), but none are found in the drier forests of New

͸ͺʹ

Caledonia (Barrabé et al., 2014).

͸ͺ͵ ͸ͺͶ

5. Conclusions The present study on Pacific Cyrtandra has provided detailed estimates of historical

͸ͺͷ ͸ͺ͸

biogeography both among and within archipelagos, improved phylogenetic resolution, and

͸ͺ͹

refined estimates of divergence times. However, the current phylogenetic framework based on

͸ͺͺ

five molecular markers is not sufficient to decipher many evolutionary relationships within

͸ͺͻ

Pacific Cyrtandra. We suggest that even substantial additional data may well be insufficient to

͸ͻͲ

resolve fine-scale relationships (e.g., among Hawaiian species) in this recent and rapid radiation.

 

͵ͳ

͸ͻͳ

Among these recent radiations, we expect to encounter problems due to incomplete lineage

͸ͻʹ

sorting, hybridization and introgression. Coalescence-based approaches that estimate species

͸ͻ͵

trees from multi-gene sequence data will likely be necessary to resolve these issues (Liu et al.,

͸ͻͶ

2015). Increased taxon sampling in under-collected regions of Melanesia (New Guinea,

͸ͻͷ ͸ͻ͸

Bismarck Archipelago, Solomon Islands, and Vanuatu; see Table 1) and the use of

͸ͻ͹

phylogenetically informed morphological study and analysis (sensu Clark et al., 2013) will be

͸ͻͺ

most effective in further refining our understanding of the evolutionary history and species

͸ͻͻ

affinities within this genus. These poorly sampled regions of Melanesia are thought to harbor

͹ͲͲ

species that are morphologically intermediate between those found in Southeast Asia and more

͹Ͳͳ

remote Pacific islands, and could offer a more nuanced understanding of the mechanisms

͹Ͳʹ

contributing to the broad distribution and rapid diversification of Pacific Cyrtandra. Urgency

͹Ͳ͵

exists in that many species of Cyrtandra are threatened with extinction due to declines in the

͹ͲͶ

extent and quality of habitat that are associated with anthropogenic disturbance (e.g., the

͹Ͳͷ

introduction of invasive plant and animal species, deforestation, and climate change). As species

͹Ͳ͸

of Cyrtandra are lost to extinction, the pieces of this intricate puzzle are being lost and our

͹Ͳ͹

ability to infer the history of this fascinating genus is diminished. Field expeditions should not

͹Ͳͺ

only remain a priority but also be accelerated to obtain voucher specimens, tissue samples, and

͹Ͳͻ

associated information on the natural history of Cyrtandra for future study.

͹ͳͲ ͹ͳͳ

Acknowledgements We are grateful to Marika Tuiwawa, Alivereti Naikatini, Manoa Maiwaqa, Mereia Tabua,

͹ͳʹ ͹ͳ͵

and Sarah Pene (South Pacific Regional Herbarium, The University of the South Pacific) for

 

͵ʹ

͹ͳͶ

assistance in Fiji. We also thank David Lorence, Ken Wood and Tim Flynn (National Tropical

͹ͳͷ

Botanical Garden), as well as Talie Foliga, Moeumu Uili, Fialelei Enoka and Josef Pisi (Samoa

͹ͳ͸

Ministry of Natural Resources and Environment) for assistance in Samoa. The Fiji Ministry of

͹ͳ͹

Education, Samoa Ministry of Natural Resources and Environment, Fiji Biosecurity, and the

͹ͳͺ

United States Department of Agriculture granted permits. We thank the following people for

͹ͳͻ

providing DNA samples: Hannah Atkins (Royal Botanic Gardens Edinburgh); Elizabeth Stacy

͹ʹͲ

(The University of Hawai‘i at Hilo); Yohan Pillon (University of Limoges, France); Gemma

͹ʹͳ

Bramley (Royal Botanic Gardens Kew); Eric Roalson (University of Washington); Jean-Yves

͹ʹʹ

Meyer (French Polynesian Research Department); and David Lorence. M.A.J. thanks Gregory

͹ʹ͵

Hora for assistance in the field and moral support; J. Travis Columbus for advice and assistance

͹ʹͶ

with import permits; Sandra Namoff, Carrie Kiel, and Diana Jolles for advice and assistance in

͹ʹͷ

the lab; Gabe Johnson and Carol Kelloff for generating outgroup sequences; Nick Matzke for

͹ʹ͸

advice on BioGeoBEARS analyses; Eric Roalson and J. Mark Porter for advice on divergence

͹ʹ͹

time analyses; the local people of Fiji and Samoa for their assistance, expertise and hospitality.

͹ʹͺ

The following herbaria kindly allowed the use of their collections for study: BISH, GH, K, NY,

͹ʹͻ

RSA, SUVA, UC, US, and WU. Funding for this project was provided by the following: Rancho

͹͵Ͳ

Santa Ana Botanic Garden; Garden Club of America Award in Tropical Botany; American

͹͵ͳ

Philosophical Society Lewis and Clark Fund for Exploration and Field Research; The Gesneriad

͹͵ʹ

Society Nelly D. Sleeth Scholarship Endowment Fund; The Gesneriad Society Elvin McDonald

͹͵͵

Fund; Society of Systematic Biologists Graduate Student Research Award; Sigma Xi Grants-in-

͹͵Ͷ

Aid of Research; American Society of Plant Taxonomists Graduate Student Research Grant;

͹͵ͷ

Claremont Graduate University; and The Smithsonian Institution.

͹͵͸

 

͵͵

͹͵͹

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Ͷ͵

Figure 1. Maximum likelihood tree from the concatenated partitioned analysis with branches color-coded for each geographic region (see map in Figure 3). Node support is indicated as maximum likelihood bootstrap support (BS) and Bayesian posterior probabilities (PP). An asterisk indicates 100% BS or 1.0 PP; a dash indicates that the branch was not supported (< 50% BS or < 0.50 PP values). Photographs depict examples of fruit morphology in Pacific Cyrtandra, with boxes color-coded by geographic region.

Figure 2. Maximum clade credibility tree of Cyrtandra based on a BEAST analysis of five concatenated loci (nrITS, nrETS, Cyrt1, psbA-trnH, and rpl32-trnL). Mean divergence time estimates are shown as millions of years ago (Mya) with 95% highest posterior density (blue boxes). Stars indicate calibration points: 1) crown age of Cyrtandra = 11.1 mya, 2) crown age of the Pacific clade of Cyrtandra = 6.4 mya, and 3) maximum geologic age of the main Hawaiian Islands = 4.7 mya. Branches are color-coded by geographic region as in other Figures.

Figure 3. Ancestral range estimation for Cyrtandra based on the chronogram produced in BEAST, and the best model determined in BioGeoBEARS (BayArea-like+j+x). Areas are colorcoded for 16 geographic regions depicted in the map. Pie graphs at each node indicate the probability of a given area (or combined areas). Arrows on the map depict the direction of founder events between islands as determined in a biogeographic stochastic mapping analysis.

Figure 4. Ancestral range estimation for Hawaiian Cyrtandra based on the chronogram produced in BEAST, and the best model determined in BioGeoBEARS (BayArea-like+j+x). Areas are color-coded for the four main Hawaiian Islands depicted in the map. Pie graphs at each

 

ͶͶ

node indicate the probability of a given area (or combined areas). Arrows on the map depict the direction and number of founder events between islands as determined in a biogeographic stochastic mapping analysis.

 

Ͷͷ

Table 1. Taxon list for 163 individuals sampled in the current study including five outgroup species (Aeschynanthus spp. and Agalmyla spp.). Species are listed in alphabetical order. GenBank accession numbers are included for each of the five genic regions used in the current study. The * symbol indicates unsequenced regions. Species

Origin

Collector & No.; Institution

ITS

ETS

Cyrt1

psbA-trnH

Aeschynanthus longicaulis Wall ex. R. Brown Aeschynanthus solomonensis P. Woods Aeschynanthus tricolor Hook. Agalmyla bilirana Hilliard & B.L. Burtt Agalmyla glabra (Copel ex. Merr.) Hilliard & B.L. Burtt Cyrtandra aloisiana A.C. Sm. C. anthropophagorum ex. A. Gray C. atherocalyx C. aurantiicarpa G.W. Gillett C. baileyi F. Muell. in Bailey, F.M. C. aff. bidwillii C.B. Clarke C. biserrata H. St. John C. calpidicarpa H. St. John & Storey C. cephalophora Gillespie C. cephalophora Gillespie C. chlorantha A.C. Sm. C. chlorantha A.C. Sm. C. ciliata Seem. C. ciliata Seem. C. clarkei Stapf C. coccinea Blume C. coleoides Seem. C. coleoides Seem. C. compressa C.B. Clarke C. cordifolia Gaudich. C. dolichocarpa A. Gray C. dolichocarpa A. Gray C. efatensis Guillaumin C. cf. elata Schltr. C. elizabethae H. St. John C. erectiloba G.W. Gillett C. erectiloba G.W. Gillett

Indonesia Solomon Islands, Isabel Indonesia Philippines, Leyte Philippines, Camiguin Is.

MSBG 1974-2207-W Clark 725; PTBG MSBG 1974-1760-W RBGE-PNHE 19991910A RBGE-PNHE 19991924A

EU919959

EU919898

EU920018

EU919958

EU919897

* * * * *

Fiji, Viti Levu Fiji, Viti Levu Solomon Islands, Isabel Samoa, Savaii Australia, Queensland Society Islands, Hua Hine Hawaii, Molokai Hawaii, Oahu Fiji, Viti Levu Fiji, Vanua Levu Fiji, Viti Levu Fiji, Viti Levu Fiji, Taveuni Fiji, Taveuni Borneo, Sabah Indonesia, Java Fiji, Viti Levu Fiji, Viti Levu Samoa, Savaii Hawaii, Oahu Fiji, Vanua Levu Fiji, Vanua Levu Vanuatu, Espiritu Santo Papua New Guinea, Morobe Austral Islands, Rurutu Solomon Islands, Isabel Bismarck Archipelago, New Britain Papua New Guinea, Morobe Papua New Guinea, Morobe Papua New Guinea, Central Fiji, Viti Levu Fiji, Viti Levu Samoa, Upolu Samoa, Savaii Marquesas, Hiva Oa

Johnson 113; RSA Johnson 115; SUVA Clark 729; PTBG Clark 655; PTBG Costion 1687; CNS Wood 11072; PTBG Wood 11386; PTBG Clark 571; PTBG Johnson 01; RSA Johnson 44; SUVA Johnson 141; RSA Johnson 142; SUVA Johnson 93; RSA Johnson 210; RSA Cronk et al. 19; RBGE Hoover & Agus ARs 167; US Johnson 132; RSA Johnson 134; SUVA Clark 652; PTBG Clark 579; PTBG Johnson 40; SUVA Johnson 53; RSA Pillon 536; NOU James 0444; BISH Meyer, photo voucher; RSA Clark 745; E, K, PTBG, US James 0134; BISH

EU919971 KF148662 GQ475176 GQ475194 EU919951

EU919910 KF148650 GQ475089 GQ475107 EU919890

EU919972

EU919911

GQ475131

EU919970 EU919955

EU919909 EU919894

EU920029 EU920014

C. erectiloba G.W. Gillett C. erectiloba G.W. Gillett C. erectiloba G.W. Gillett C. esothrix A.C. Sm. C. esothrix A.C. Sm. C. falcifolia C.B. Clarke C. falcifolia C.B. Clarke C. feaniana F.Br.

 

James 0378; BISH James 0586; BISH James 1432; BISH Johnson 10; SUVA Johnson 38; RSA Kiehn 940823-4/3; WU Wood 16973; PTBG Price 200; PTBG

Ͷ͸

*

KF148652

*

EU920017 *

EU920030 KF148674 GQ475139 GQ475157 EU920010

* * KF148640

KF148664 *

*

* *

GQ475184

GQ475097

EU919960

EU919899

*

rpl32-trnL

GQ475147 EU920019

* *

C. filibracteata B.L. Burtt C. fulvovillosa Rech. C. futunae Kraenzl. C. giffardii Rock C. graeffei C.B. Clarke C. grandiflora Gaudich. C. grayana Hillebr. C. grayi C.B. Clarke C. hashimotoi Rock C. hawaiensis C.B. Clarke C. hawaiensis C.B. Clarke C. hawaiensis C.B. Clarke C. hawaiensis C.B. Clarke C. heinrichii H. St. John C. hirtigera var. hirtigera H.J. Atkins & Cronk C. hornei C.B. Clarke C. induta A. Gray C. involucrata Seem. C. jonesii (F.Br.) G.W. Gillett C. jugalis A.C. Sm. C. cf. kandavuensis A.C. Sm. C. kauaiensis Wawra C. kaulantha H. St. John & Storey C. kealiae Wawra subsp. urceolata W.L. Wagner & Lorence C. kenwoodii W.L. Wagner & A.J. Wagner C. cf. kohalae Rock C. kruegeri Rein. C. kusaimontana Hosok. C. laxiflora H. Mann C. leucantha A.C. Sm. C. leucantha A.C. Sm. C. leucantha A.C. Sm. C. longifolia (Wawra) Hillebr. ex. C.B. Clarke C. lydgatei Hillebr. C. lysiosepala C.B. Clarke C. macrocalyx Hillebr. C. macrotricha G.W. Gillett C. mareensis Däniker C. menziesii Hook. & Arn. C. mesilauensis B.L. Burtt C. milnei Seem. ex. A. Gray C. milnei Seem. ex. A. Gray C. milnei Seem. ex. A. Gray C. multiseptata Gillespie C. multiseptata Gillespie C. munroi C.N. Forbes C. munroi C.N. Forbes

 

Solomon Islands, Kolombangara Solomon Islands, Kolombangara Wallis and Futuna, Alofi Hawaii, Hawaii Island Samoa, Upolu Hawaii, Oahu Hawaii, Maui Hawaii, Maui Hawaii, Maui Hawaii, Oahu Hawaii, Maui Hawaii, Hawaii Island Hawaii, Molokai Hawaii, Kauai Philippines, Palawan Fiji, Viti Levu Society Islands, Tahiti Fiji, Viti Levu Marquesas, Ua Huka Fiji, Viti Levu Fiji: Kadavu Hawaii, Kauai Hawaii, Oahu Hawaii, Kauai

Clark 770; E, K, PTBG, US Clark 785; E, K, PTBG, US Meyer, photo voucher, RSA Johansen 31; RSA Wood 16898; PTBG Clark 577; PTBG Clark 666; PTBG Clark 676; PTBG Oppenheimer H80403; BISH Clark 569; PTBG Clark 661; PTBG Johansen, photo voucher; RSA Wood 11391; PTBG Johansen, photo voucher; RSA Cronk et al. 25433; RBGE Johnson 135; RSA, SUVA Meyer, photo voucher; RSA Johnson 199; RSA, SUVA Wood 10484; PTBG Johnson 147; RSA Johnson 203; RSA Clark 558; PTBG Clark 572; PTBG Perlman 18805; PTBG

KF148658

Marquesas, Ua Pou Hawaii, Hawaii Samoa, Upolu Micronesia, Kosrae Hawaii, Oahu Fiji, Taveuni Fiji, Viti Levu Fiji, Taveuni Hawaii, Kauai Hawaii, Maui Hawaii, Hawaii Island Hawaii, Molokai Solomon Islands, Isabel Loyalty Islands, Lifou Hawaii, Hawaii Island Borneo, Sabah Fiji, Viti Levu Fiji, Viti Levu Fiji, Kadavu Fiji, Viti Levu Fiji, Viti Levu Hawaii, Maui Hawaii, Lanai

Wood 10804; PTBG Johansen, photo voucher; RSA Wood 16853; PTBG Flynn 5995; PTBG Clark 568; PTBG Johnson 81; RSA Johnson 197; RSA Johnson 214; RSA Johnson 224; RSA Oppenheimer H120809; BISH Johnson 222; RSA Oppenheimer H110622; BISH Clark 747; PTBG Butaud 3361; NOU Johansen, photo voucher; RSA Cronk et al. 6; RBGE Johnson 28; RSA Johnson 194; RSA Johnson 202; RSA Johnson 128; RSA Johnson 170; RSA, SUVA Clark 675; PTBG Oppenheimer H120638; BISH

Ͷ͹

KF148646

KF148670

* EU919954 EU919982 EU919984

EU919893 EU919921 EU919923

EU919949

EU919888

*

EU920013 EU920039 EU920040

* *

* EU920008 * *

EU919965

EU919904

EU920024

GQ475167 EU919952 EU919957

GQ475079 EU919891 EU919896

GQ475121 EU920011 EU920016

GQ475174

GQ475087

EU919945 EU919948

EU919884 EU919887

EU920004 EU920007

GQ475190 KF148653

GQ475103 KF148641

GQ475153 KF148665

*

*

GQ475130 *

*

*

EU919983

EU919922

GQ475134 *

C. nanawalensis H. St. John C. nitens C.B. Clarke C. occulta A.C. Sm. C. occulta A.C. Sm. C. ootensis F. Br. C. oxybapha W.L. Wagner & D.L. Herbst C. pachyneura Kraenzl. C. paludosa Gaud. var. microcarpa Wawra C. paludosa Gaud. var. paludosa C. paludosa Gaud. var. paludosa C. paludosa Gaud. var. paludosa C. peltata Jack C. pendula Blume C. pickeringii A. Gray C. platyphylla A. Gray C. platyphylla A. Gray C. pogonantha A. Gray C. pogonantha A. Gray C. polyantha C.B. Clarke C. prattii Gillespie C. prattii Gillespie C. pritchardii Seem. C. pritchardii Seem. C. procera Hillebr. C. propinqua C. Forbes C. pulchella Rich ex. A. Gray C. richii A. Gray C. richii A. Gray C. roseiflora H.J. Atkins C. samoensis A. Gray C. samoensis A. Gray C. samoensis A. Gray C. sandwicensis A. Gray C. schizocalyx G.W. Gillett C. sessilis H. St. John & Storey C. sp. ‘kadavu’ C. sp. ‘hua hine’ C. sp. ‘isabel 1’ C. sp. ‘isabel 2’ C. sp. ‘kolombangara’ C. sp. ‘vangunu’ C. sp. ‘espiritu santo’ C. sp. ‘hiu’ C. sp. ‘morobe 1’ C. sp. ‘morobe 2’ C. sp. ‘central’ C. sp. nov. 1 C. sp. nov. 2

 

Hawaii, Hawaii Island Samoa, Savaii Fiji, Viti Levu Fiji, Viti Levu Marquesas, Hiva Oa Hawaii, Maui Philippines, Luzon Hawaii, Kauai Hawaii, Hawaii Island Hawaii, Oahu Hawaii, Maui Indonesia, Sumatra Malaysia, Selangor Hawaii, Kauai Hawaii, Hawaii Island Hawaii, Maui Samoa, Upolu Samoa, Savaii Hawaii, Oahu Fiji, Viti Levu Fiji, Viti Levu Fiji, Viti Levu Fiji, Viti Levu Hawaii, Maui Hawaii, Oahu Samoa, Tau Samoa, Savaii Samoa, Upolu Indonesia, Sulawesi Samoa, Ofu Tonga, Tofua Samoa, Upolu Hawaii, Oahu Vanuatu, Espiritu Santo Hawaii, Oahu Fiji, Kadavu Society Islands, Hua Hine Solomon Islands, Isabel Solomon Islands, Isabel Solomon Islands, Kolombangara Solomon Islands, Vangunu Vanuatu, Espiritu Santo Vanuatu, Torres Islands, Hiu Papua New Guinea, Morobe Papua New Guinea, Morobe Papua New Guinea, Central Fiji, Taveuni Fiji, Taveuni

Johansen 11; RSA Wood 17015; PTBG Plunkett 1838; US Clark 694; PTBG Wood 10047; PTBG Oppenheimer H100511; BISH Wen 8271; RBGE Johnson, photo voucher; RSA Johnson 223; RSA Johansen 36; RSA Oppenheimer H40408; BISH Radhiah & Cronk 71; RBGE Bramley GB37; RBGE Lorence 9528; PTBG Johansen 28; RSA Oppenheimer H100512; BISH Clark 649; PTBG Wood 16941; PTBG Johansen, photo voucher, RSA Johnson 164; RSA Johnson 165; RSA Johnson 18; RSA Johnson 37; SUVA Oppenheimer H110621; BISH Clark 570; PTBG Lorence 8525; PTBG Clark 650; PTBG Wood 16892; PTBG Mendum et al. 173; RBGE Lorence 8633; PTBG RP 71221; PTBG Kiehn 940819-1/1; WU Clark 576; PTBG Pillon 534; NOU Roalson 1577-07; WS Johnson 204; RSA Wood 11057; PTBG Clark 724; PTBG Clark 726; PTBG Clark 787; PTBG Clark 795; PTBG Pillon 535; NOU Pillon 1082; NOU James 0535; BISH James 0809; BISH James 1232; BISH Johnson 91; RSA Johnson 103; RSA

Ͷͺ

* EU919933 EU919989 EU919961

EU919872 EU919928 EU919900

GQ475179

GQ475092

GQ475142

GQ475191 EU919968

GQ475104 EU919907

GQ475154 EU920027

GQ475189 EU919950 EU919941 EU919969

GQ475102 EU919889 EU919880 GQ475083

GQ475152 EU920009 EU920000 EU920028

*

EU919992 GQ475137 EU920020

*

* EU919942 EU919943 GQ475185 EU919953

EU919881 EU919882 GQ475098 EU919892

EU920001 GQ475122 GQ475148 EU920012

GQ475199

GQ475112

GQ475162

GQ475177

GQ475090

*

GQ475140

KF148659

KF148647

*

KF148671

*

* * * *

* *

C. sp. nov. 3 C. sp. nov. 4 C. sp. nov. 4 C. sp. nov. 5 C. spathacea A.C. Sm. C. spathacea A.C. Sm. C. spathulata H. St. John C. subulibractea G.W. Gillett C. subulibractea G.W. Gillett c. subumbellata H. St. John & Storey C. sulcata Blume C. tahuatensis Fosberg & Satchet C. taviunensis Gillespie C. tempestii Horne ex. C.B. Clarke C. tempestii Horne ex. C.B. Clarke C. terrae-guilelmi K. Schum. C. thibaultii Fosberg & Satchet C. tintinnabula Rock C. tomentosa A.C. Sm. C. urvillei C.B. Clarke C. victoriae Gillespie C. victoriae Gillespie C. vitiensis Seem. C. vitiensis Seem. C. wagneri Lorence & Perlman C. waianaeensis H. St. John & Storey C. wainihaensis H. Lév. C. wawrae C.B. Clarke

Fiji, Taveuni Fiji, Vanua Levu Fiji, Vanua Levu Fiji, Vanua Levu Fiji, Kadavu Fiji, Kadavu Hawaii, Maui Solomon Islands, Vangunu Papua New Guinea, Morobe Hawaii, Oahu Indonesia, Java Marquesas, Tahuata Fiji, Taveuni Fiji, Taveuni Fiji, Taveuni Papua New Guinea, Morobe Marquesas, Ua Pou Hawaii, Hawaii Island Fiji, Viti Levu Micronesia, Kosrae Fiji, Viti Levu Fiji, Viti Levu Fiji, Viti Levu Fiji, Viti Levu Hawaii, Hawaii Island Hawaii, Oahu Hawaii, Kauai Hawaii, Kauai

Johnson 105; RSA Johnson 61; RSA Johnson 59; SUVA Johnson 49; SUVA Johnson 206; RSA Johnson 208; RSA Clark 664; PTBG Clark 796; K, PTBG James 0445; BISH Johansen, photo voucher; RSA Hoover & Agus ARs 160; US Wood 6563; PTBG Johnson 220; RSA Johnson 82; RSA Johnson 83; RSA James 0376; BISH Wood 10428; PTBG Perlman 17676; PTBG Johnson 153; RSA Lorence 7838; PTBG Johnson 139; RSA, SUVA Johnson 140; RSA Johnson 07; RSA Johnson 114; RSA Lorence 8907; PTBG Johansen, photo voucher; RSA Clark 549; PTBG Clark 550; PTBG



 

Ͷͻ

*

EU919981

EU919920 *

EU920038 *

EU919980 GQ475178

EU919919 GQ475091

GQ475132 GQ475141

EU919966 EU919930

EU919905 EU919869

* EU920025 GQ475114

EU919946

EU919885

EU920005

GQ475166

GQ475078

*

EU919937 EU919938

EU919876 EU919877

GQ475116 * *

EU919996 EU919997

 

ͷͲ

Table 2. Current estimate of species numbers in Cyrtandra by geographic region (Atkins et al., 2013), and the number of species included in the present study. The order in which regions are presented is from a general west to east direction (i.e., Southeast Asia, Melanesia, Polynesia). Geographic Region

No. of species

No. sampled

Nicobar Islands Thailand Peninsular Malaysia Sumatra Java Lesser Sunda Islands Borneo Taiwan Philippines Sulawesi Moluccas New Guinea Bismarck Archipelago Solomon Islands Australia Loyalty Islands Vanuatu Micronesia Wallis and Futuna Fiji Samoa Tonga Cook Islands Society Islands Austral Islands Marquesas Hawaii

2 6 9 40–44 19–32 3 181–200 1 105–150 22–40 3 107–120 12–18 16–20 1 1 10–13 2–3 1 37–46 20–22 1 2 16 1 11 60

0 0 1 1 2 0 2 0 2 1 0 7 1 8 1 1 4 2 1 30 10 1 0 3 1 6 38

 

ͷͳ

Table 3. Estimated ages (mya) of select major nodes in the present study using BEAST, and comparable node dates from earlier studies. Node dates that were not reported are termed N/A. Ranges in parentheses represent the 95% highest posterior density (HPD). Node Cyrtandra stem Cyrtandra crown Pacific Cyrtandra stem Pacific Cyrtandra crown Fiji B crown Samoa A crown Samoa B/C/D crown Marquesas crown

 

BEAST age (present study) 25.57 (16.90–35.02) 17.29 (12.54–22.15) 9.79 (7.41–12.38) 8.63 (6.58–10.70) 2.73 (1.25–4.43) 2.48 (1.31–3.66) 3.51 (2.40–4.67) 2.54 (1.42–3.79)

BEAST age (Roalson and Roberts, 2016) 30.45 (14.79–37.54) 11.08 (3.25–21.78) N/A 6.29 (3.65–9.50) N/A N/A N/A N/A

ͷʹ

r8s age (Clark et al., 2009) N/A 48 39.2 (36.5–41.9) 21.7 (17.0–26.4) 8.9 (4.5–13.3) 11.6 (7.1–16.1) 12.0 (7.7–16.3) 5.8 (5.3–6.3)

Table 4. Event counts for Pacific and Hawaiian Cyrtandra from 500 biogeographic stochastic mappings in BioGeoBEARS. Mapping was performed using parameters from the best model of biogeography: BayArea-like+j+x for Pacific Cyrtandra and BayArea-like+j+x for Hawaiian Cyrtandra. The BayArea-like+j+x model does not incorporate subset sympatry or vicariance, which is indicated by N/A. Biogeographical Event Range switching Range expansion Narrow sympatry Widespread sympatry Subset sympatry Vicariance Founder event Total events

 

Pacific Mean 0 0 0 0 N/A N/A 24.75 24.75

Pacific SD 0 0 0 0 N/A N/A 0.84 0.84

ͷ͵

Hawaii Mean 0 0 0 0 N/A N/A 10.18 10.18

Hawaii SD 0 0 0 0 N/A N/A 1.01 1.01

*Graphical Abstract (for review) Hawaii

Southeast Asia

Micronesia

New Guinea Bismarck Archipelago Solomon Islands Marquesas Vanuatu

Wallis & Futuna Society Islands Samoa

Australia Loyalty Islands

Fiji

Tonga

New Zealand

Austral Islands

Figure 1a C. tintinnabula Hawaii, Hawai’i Island C. nanawalensis Hawaii, Hawai’i Island C. hawaiensis Hawaii, Hawai’i Island C. cf. kohalae Hawaii, Hawai’i Island - /58 C. menziesii Hawaii, Hawai’i Island C. platyphylla Hawaii, Hawai’i Island 85/99 1 C. biserrata Hawaii, Moloka’i - /89 C. macrocalyx Hawaii, Moloka’i C. procera Hawaii, Maui 92/ * C. grayi Hawaii, Maui 90/ * C. grayana Hawaii, Maui C. hashimotoi Hawaii, Maui - /96 C. spathulata Hawaii, Maui 80/66 C. lydgatei Hawaii, Maui C. paludosa var. microcarpa Hawaii, Kaua’i C. munroi Hawaii, Lana’i 92/ * C. hawaiensis Hawaii, Moloka’i 9 - /67 C. lysiosepala Hawaii, Hawai’i Island C. platyphylla Hawaii, Maui C. wagneri Hawaii, Hawai’i Island 0.02 substitutions per site C. giffardii Hawaii, Hawai’i Island 63/98 C. munroi Hawaii, Maui C. oxybapha Hawaii, Maui - /86 C. cordifolia Hawaii, O’ahu C. propinqua Hawaii, O’ahu C. subumbellata Hawaii, O’ahu - /79 C. laxiflora Hawaii, O’ahu C. sandwicensis Hawaii, O’ahu C. waianaeensis Hawaii, O’ahu C. polyantha Hawaii, O’ahu C. paludosa var. paludosa Hawaii, O’ahu 53/ * 87/ * C. kaulantha Hawaii, O’ahu C. hawaiensis Hawaii, O’ahu 90/ * - /72 C. calpidicarpa Hawaii, O’ahu 99/ * 8 70/ * C. sessilis Hawaii, O’ahu - /63 C. grandiflora Hawaii, O’ahu C. hawaiensis Hawaii, Maui */ * C. kauaiensis Hawaii, Kaua’i 21 67/94 */ * C. wainihaensis Hawaii, Kaua’i - /67 C. kealiae ssp. urceolata Hawaii, Kaua’i - /89 C. wawrae Hawaii, Kaua’i 67/ * C. paludosa var. paludosa Hawaii, Maui C. paludosa var. paludosa Hawaii, Hawai’i Island 92/ * 20 C. pickeringii Hawaii, Kaua’i */ * - /56 7 C. heinrichii Hawaii, Kaua’i 53/98 C. longifolia Hawaii, Kaua’i C. occulta Fiji, Viti Levu 96/ * */ * C. occulta Fiji, Viti Levu C. tomentosa Fiji, Viti Levu 19 C. victoriae Fiji, Viti Levu */ * 92/ * 6 * / * C. victoriae Fiji, Viti Levu C. taviunensis Fiji, Taveuni Cyrtandra sp. ‘isabel 2’ Solomon Islands, Isabel 96/ * Cyrtandra sp. ‘isabel 1’ Solomon Islands, Isabel */ * 18 Cyrtandra sp. ‘vangunu’ Solomon Islands, Vangunu 85/ * 5 Cyrtandra sp. ‘kolombangara’ Solomon Islands, Kolombangara 85/ * Cyrtandra sp. ‘morobe 1’ Papua New Guinea, Morobe C. sulcata Java 99/ * 97/ * 99/ * C. coccinea Java 4 */ * C. pachyneura Philippines 17 C. roseiflora Sulawesi C. hirtigera var. hirtigera Philippines 16 C. atherocalyx Solomon Islands, Isabel 87/ * 98/ * C. erectiloba Bismarck Archipelago, New Britain C. erectiloba Solomon Islands, Isabel 95/99 98/ * C. baileyi Australia */ * 3 C. efatensis Vanuatu, Espiritu Santo 98/* 60/C. erectiloba Papua New Guinea, Morobe */ * C. subulibractea Papua New Guinea, Morobe C. erectiloba Papua New Guinea, Morobe 81/96 C. erectiloba Papua New Guinea, Central 61/95 Cyrtandra sp. ‘central’ Papua New Guinea, Central C. subulibractea Solomon Islands, Vangunu 99/ * */ * */ * 2 */ * 15 Cyrtandra sp. ‘espiritu santo’ Vanuatu, Espiritu Santo 68/84 C. filibracteata Solomon Islands, Kolombangara C. terrae-guilelmi Papua New Guinea, Morobe Cyrtandra sp. ‘morobe 2’ Papua New Guinea, Morobe 85/ * C. cf. elata Papua New Guinea, Morobe 1 C. peltata Sumatra */ * */ * C. pendula Malaysia C. clarkei Borneo 14 84/ * C. mesilauensis Borneo - /65

Pacific clade

*/ -

Agalmyla glabra

*/ *

Agalmyla bilirana */ * */ *

Aeschynanthus tricolor Aeschynanthus solomonensis Aeschynanthus longicaulis

Figure 1b Click here to download high resolution image

Figure 2 Click here to download high resolution image

Figure 3

C. mesilauensis C. clarkei C. pendula C. peltata C. cf. elata C. terrae-guilelmi C. sp. ‘morobe 2’ C. filibracteata C. subulibractea C. sp. ‘espiritu santo’ C. sp. ‘central’ C. subulibractea C. erectiloba C. erectiloba C. erectiloba C. efatensis C. baileyi C. hirtigera var. hirtigera C. atherocalyx C. roseiflora C. pachyneura C. sulcata C. coccinea C. sp. ‘morobe 1’ C. sp. ‘kolombangara’ C. sp. ‘isabel 1’ C. taviunensis C. victoriae C. tomentosa C. occulta C. hawaiensis C. grandiflora C. kaulantha C. hawaiensis C. sessilis C. calpidicarpa C. longifolia C. pickeringii C. heinrichii C. paludosa var. paludosa C. paludosa var. paludosa C. wawrae C. kealiae ssp. urceolata C. wainihaensis C. kauaiensis C. propinqua C. paludosa var. paludosa C. waianaeensis C. polyantha C. sandwicensis C. laxiflora C. cordifolia C. oxybapha C. munroi C. subumbellata C. platyphylla C. lysiosepala C. wagneri C. giffardii C. macrocalyx C. biserrata C. platyphylla C. menziesii C. tintinnabula C. nanawalensis C. hawaiensis C. cf. kohalae C. procera C. grayi C. grayana C. munroi C. paludosa var. microcarpa C. hawaiensis C. lydgatei C. spathulata C. hashimotoi C. macrotricha C. fulvovillosa C. pogonantha C. richii C. pogonantha C. richii C. aurantiicarpa C. thibaultii C. jonesii C. kenwoodii C. tahuatensis C. ootensis C. feaniana C. tempestii C. schizocalyx C. mareensis C. samoensis C. samoensis C. futunae C. sp. ‘hiu’ C. samoensis C. urvillei C. kusaimontana C. sp. nov. 3 C. sp. ‘hua hine’ C. nitens C. pulchella C. compressa C. kruegeri C. graeffei C. falcifolia C. falcifolia C. sp. nov. 2 C. ciliata C. sp. nov. 1 C. cephalophora C. cephalophora C. sp. nov. 5 C. sp. nov. 4 C. dolichocarpa C. aff. bidwillii C. induta C. elizabethae C. jugalis C. pritchardii C. involucrata C. aloisiana C. anthropophagorum C. esothrix C. prattii C. sp. ‘kadavu’ C. multiseptata C. hornei C. coleoides C. cf. kandavuensis C. spathacea C. leucantha C. chlorantha C. vitiensis C. leucantha C. milnei C. milnei

Southeast Asia/New Guinea Southeast Asia/Solomon Islands Southeast Asia New Guinea Bismarck Archipelago Solomon Islands Vanuatu Australia Fiji Hawaii Samoa Marquesas Loyalty Islands Society Islands Tonga Wallis & Futuna Micronesia Austral Islands

Hawaii

Southeast Asia

Micronesia

New Guinea Bismarck Archipelago Solomon Islands Marquesas anuatu Vanuatu

W allis & Futuna Wallis Society Islands Samoa

Australia Loyalty Islands

Fiji

Tonga Austral Islands

New Zealand

15

10

5

0 Mya

Figure 4 M C. hawaiensis (Maui) O C. grandiflora O C. kaulantha O C. hawaiensis O C. sessilis O C. calpidicarpa K C. longifolia K C. pickeringii

KO 23%

K C. heinrichii H C. paludosa var. paludosa M C. paludosa var. paludosa K C. wawrae K C. kealiae ssp. urceolata K C. wainihaensis

KO 32%

K C. kauaiensis O C. propinqua O C. paludosa var. paludosa O C. waianaeensis O C. polyantha O C. sandwicensis O C. laxiflora O C. cordifolia M C. oxybapha M C. munroi (Maui) O C. subumbellata M C. platyphylla H C. lysiosepala H C. wagneri H C. giffardii M C. macrocalyx M C. biserrata H C. platyphylla H C. menziesii H C. tintinnabula H C. nanawalensis H C. hawaiensis

1

H C. cf. kohalae 1 1

M C. procera M C. grayi

2

M C. grayana

2

M C. munroi (Lana’i)

3

K Kaua‘i

K C. paludosa var. microcarpa

O O‘ahu

M C. hawaiensis (Moloka’i)

M Maui Nui

M C. lydgatei M C. spathulata

H Hawai‘i

M C. hashimotoi

4

3

2

1

0 Mya