Phylogenies and Chromosome Evolution of Phacelia (Boraginaceae: Hydrophylloideae) Inferred from Nuclear Ribosomal and Chloroplast Sequence Data

2014·39.9 MB·English

by  Genevieve K Walden

Madrono, Vol. 61, No. 1, pp. 16-47, 2014
PHYLOGENIES AND CHROMOSOME EVOLUTION OF PHACELIA
(BORAGINACEAE: HYDROPHYLLOIDEAE) INFERRED FROM NUCLEAR
RIBOSOMAL AND CHLOROPLAST SEQUENCE DATA
Genevieve K. Walden
CA
Department of Integrative Biology, University of California, Berkeley, 94720
gkwalden@gmaiLcom
Laura M. Garrison
Department ofEcology and Evolutionary Biology, Brown University, Providence, RI 02912
Greg S. Spicer, Frank W. Cipriano, and Robert Patterson
Department of Biology, San Francisco State University, 1600 Holloway Avenue,
CA
San Francisco, 94132
Abstract
This project sampled throughout Phaceliausingtheinternal transcribed spacerregion (ITS-l, ITS-
DNA DNA
2, and 5.8S gene) ofnuclear ribosomal (nrlTS) and the chloroplast gene {ndhV) to infer
phytogenies fornuclearand plastid partitions. Nuclearand plastid partitionswere incongruentin our
analyses. Phylogenetic analyses (maximum parsimony, maximumlikelihood, and Bayesian inference)
recovered gene tree topologies similar to previous molecular studies. We corroborate incongruence
between nuclear and plastid topologies for placement of some problematic groups (e.g., Draperia,
Romanzoffia and “core” Phacelia subg. Pulchellae, Phacelia sect. Baretiana). Combined analyses
resultedinbetterresolutionthanseparateanalyses, andinatopologythatfavoredtheseparateplastid
topologies. Romanzojfia was sister to a monophyletic Phacelia in the combined analyses. Our results
support combining incongruent partitions in a combined analysis to seek support for internal nodes.
Maximum likelihood analyses were used to infer ancestral chromosome numbers and identify gains,
losses, polyploiddoubling, andwholegenomeduplicationevents frompublishedchromosomecounts
in the genus. The predicted base number for the genus was x = 9, x = 11, or x = 12.
KeyWords: Boraginaceae, Californiaflora,chromosomeevolution, Hydrophylloideae,incongruence,
molecular phylogenetics, Phacelia, Romanzoffia.
Phacelia Juss. is the largest genus (207 spp.) in example of the diversity of the California flora
Hydrophylloideae (Boraginaceae). The majority (Stebbins and Major 1965; Raven and Axelrod
of species (176 spp.) are distributed in western 1978; Ackerly 2009; Kraft et al. 2010). However,
North America and an additional 16 species research regarding evolution and diversification
occur into Central America, with an amphitropi- in the genus and its significance in the California
cal disjunct group ofnine species in southwestern flora has been limited due to the lack of a well-
South America. The center of diversity for the resolved, broadly sampled molecular phylogeny
genus is the California Floristic Province (CFP); with congruent nuclear and plastid partitions.
a third of described taxa occur within the CFP
(ca. 70 spp., 40 spp. endemic) and ca. 90 spp. Recent Molecular Studies
o20c1c1u)r within the political boundaries ofthe state
(Raven and Axelrod 1978; Patterson et al. 2012). Relationships of major lineages in Phacelia
In California, 33 taxa in Phacelia are ranked in have been previously studied using molecular
t2h0e12)California Native Plant Society Rare and phylogenetic methods. Gilbert et al. (2005)
Endangered Plant Inventory (CNPS 2011). These combined thesis work in Phacelia sect. Euglypta
include one ofthree federallyendangered taxa {P. S. Watson by Dempcy (1996) and in Phacelia
insularis Munz var. insularis) and one candidate sect. Miltitzia (A. de Candolle) J. T. Howell by
taxon considered for federal protection {P. Ganong (2002), along with sequences from
stellaris Brand) (U.S. Department ofthe Interior, dissertation work by Ferguson (1998), to publish
Fish and Wildlife Service 1978, 1982, 1997, 2004, an nrlTS partition of 51 taxa (84 accessions) in
. Thus, Phacelia is one often largest genera Phacelia. Gilbert et al. (2005) recovered Roman-
and Boraginaceae one of ten largest flowering zoffia Cham, sister to a monophyletic Phacelia
plant families occurring in the CFP and in and recovered a monophyletic Phacelia subg.
California (Beard et al. 2000; Baldwin et al. Microgenetes (A. de Candolle) A. Gray that
. Phacelia, as the largest and most diverse included a paraphyletic Phacelia sect. Euglypta
genus in Hydrophylloideae, is often used as an and a paraphyletic Phacelia sect, Miltitzia.
2014] WALDEN ET AL.: PHYLOGENIES AND CHROMOSOME EVOLUTION OF PHACELIA 17
Hansen et aL (2009) published an nrlTS partition analyses provide greater resolution than separate
of 56 taxa (91 accessions) and a rpll6 intron analyses of incongruent partitions (Nixon and
partition of 22 taxa (37 accessions) for Phacelia Carpenter 2005). Our goal was to determine if
sect. Gymnobytha (A, de Candolle) Benth. & increased sampling for a respective molecular
Hook.f. and Phacelia sect. Whitlavia (Harv.) marker (nrlTS, ndh¥) within Phacelia recovered
Benth. & Hook.f. Hansen et al. (2009) recovered similar gene tree topologies to previous studies
Romanzoffia sister to a monophyletic Phacelia for separate analyses of partitions, to compare
and recovered a paraphyletic Phacelia sect. nuclear and plastid partitions for character
Whitlavia in the separate nuclear partition. homogeneity and combinability (Cunningham
Phacelia was paraphyletic in the separate plastid 1997), and to determine if simultaneous analyses
partition. Both Phacelia sect. Euglypta and provided enhanced resolution for a reduced
Phacelia subg. Pulchellae (Rydb.)Walden & Patt. subset of samples for which both nrlTS and
were recovered as basal lineages with Romanzof- ndh¥ sequences were available.
fia nested within the genus. The combined nrlTS
and rpll6 intron analysis recovered Romanzoffia Chromosome Evolution
sister to a monophyletic Phacelia and monophy-
letic infrageneric sections (Hansen et al. 2009). Phacelia occupies a range of habitats and
Ferguson (1998 [1999]) included 19 taxa (19 exhibits a variety of life history traits and
accessions) of Phacelia within a larger analysis ecological adaptations. Species differences have
of Hydrophylloideae for ndhF, recovering a been traditionally based on morphological (e.g.,
paraphyletic Phacelia. Phacelia subg. Pulchellae seed shape and number) and cytological charac-
was sister to a nested Romanzoffia and remaining ters (e.g., chromosome numbers), and less so on
sampled Phacelia. ecological factors (e.g., edaphic factors). These
Collectively, researchers have published 124 characters have been used to diagnose infra- and
accessions within Phacelia using the internal interspecific taxa, and to delimit infrageneric
transcribed spacer region (ITS-1, ITS-2, and groups within the genus (for a review and current
DNA
5.8S gene) of nuclear ribosomal (nrlTS), infragenericclassification in Phacelia, see Walden
published 20 accessions for the chloroplast and Patterson [2012]). Both Constance’s (1963)
(cpDNA) gene ndh¥, and published 37 accessions and Gillett’s (1968) classifications were based
for the chloroplast (cpDNA) marker rpll6 intron largely upon chromosome numbers, drawing
(Ferguson 1998 [1999]; Olmstead et al. 2000; upon Constance’s extensive collaborations into
Gilbert et al. 2005; Hansen et al. 2009; Glass and chromosome number differences in Hydrophyl-
Levy 2011). We combined previously published laceae with Marion Cave (see Cave and Con-
nrlTS sequences with thesis work in Phacelia stance [1942, 1944, 1947, 1950, 1959]). Phacelia
sect. Glandulosae (Rydb.) Walden & R. Patt. and benefits from published chromosome counts for
Phacelia sect. Ramosissimae (Rydb) Walden & R. approximately two thirds of the genus, ranging
Patt. of Garrison (2007) in an expanded nuclear from n = 5 {P. dubia [L.] Trek & Small, P.
phylogeny to infer inter- and infraspecific evolu- maculataWood) ton = 33 (P. hastata Douglasex
tionary relationships in Phacelia. We combined Lehm. var. compacta [Greene ex Brand] Cron-
previously published ndh¥ sequences with thesis quist, P. leptosepala Rydb.) (Cave and Constance
work in Phacelia of Walden (2010) in an 1947, 1950; Kruckeberg 1956; Kovanda 1978).
expandedcpDNAphylogenyto inferinfrageneric Constance (1963); Heckard (1963), and Gillett
relationships in the genus. (1968) hypothesized that n =\\ was the ancestral
Previous molecular phylogenetic studies in condition for the genus and noted it was also the
Phacelia and Hydrophylloideae have identified most common haploid count for extant taxa.
significant phylogenetic incongruence between Hypotheses proposed forthebase numberfor the
nuclear and plastid partitions (Ferguson 1998; genus have not been tested in a broad phyloge-
Moore and Jansen 2006; Hansen et al. 2009; netic context. Previous studies considering evo-
Weeks et al. 2010; Nazaire and Hufford 2012; lution of chromosome numbers in a molecular
Taylor 2012). For an extensive discussion on context in Phacelia include mapping ofchromo-
incongruence between nuclear (nrlTS) and plas- some numbers to nrlTS tree topologies by
tid {ndh¥) partitions in Phacelia and Hydrophyl- Gilbert et al. (2005) and to ndh¥ sequence data
loideae see Ferguson (1998). When tests for using maximum parsimony by Walden (2010).
homogeneity between partitions (e.g., incongru- Reconstructing ancestral states using a maximum
ence length test [Farris et al. 1995]) reject the null parsimony approach without an explicit frame-
hypothesis, a combined analysis is inappropriate work (e.g., biosystematic studies ofchromosomal
and partitions are analyzed separately using the rearrangements across the genus) allows only for
conditional combination approach (Bull et al. coding with a categorical character matrix using
1993; Huelsenbeck et al. 1996). Some researchers the unordered states assumption in Mesquite
combine incongruent partitions using simulta- version 2.74 (Maddison and Maddison 2010),
neous analyses, arguing that these combined regardless of whether a transition represents an
MADRONO
18 [VoL 61
increasing or decreasing dysploidy event or a reference or a comprehensive list of voucher
doubling polyploid event (Mayrose et al. 2010). specimens and karyotype figures. Constance
Although the maximum parsimony approach has (1963) reported a count for Phacelia pauciflora
real merit, the unordered states assumption S. Watson without reference to a voucher
option offers little resolution for this dataset at specimen. We included this count and corre-
the present time. We were interested in determin- sponding voucher specimen examined at the
ing the ancestral base number for Phacelia to University of California Herbarium (UC) in
better understand patterns of chromosome evo- Table 1. Chromosome counts originally pub-
lution within infrageneric groups and within the lished as taxa in Phacelia but corrected or
genus using a maximum likelihood approach redetermined in later publication were excluded.
(Mayrose et al. 2010; Hallinan and Lindberg Citations are listed chronologically within each
2011a). chromEvol version 1.3 (Mayrose et al. taxon and a list of full references follows the
2010) and GDCN (Hallman and Lindberg 2011a) table. Chromosome counts were not directly
use explicit likelihood models of evolution to obtained from individuals or populations includ-
infer ancestral states for chromosome numbers at ed in the direct sequence analyses for this study.
nodes in phylogenies from rooted ultrametric This limitation may obscure cryptic diversity in
trees. These analyses offer the ability to test sampled populations. This list should be consid-
hypotheses for the base number for infrageneric ered aworkingdraft ofchromosomenumbers for
groups and the genus using results from our Phacelia and Boraginaceae. A future comprehen-
expanded nuclear and plastid phylogenies. chro- sive review of chromosome counts in Boragina-
GDCN
mEvol v.1.3 and estimate probabilities of ceae is anticipated as a useful resource for
chromosome evolution events at nodes to explore workers in the family (G. K. Walden, unpub-
patterns ofgains, losses, polyploid doubling, and lished manuscript).
whole genome duplication (WGD) events within
a known phylogeny (Mayrose et al. 2010; Taxon Sampling
Hallinan and Lindberg 2011a). Saltational sped-
ation has been an important factor in cladogen- This study represents a joint publication of
esis in the California flora; we were interested in thesis work from Garrison (2007) and Walden
identifying any recent genome duplication events (2010). The expanded nuclear partition (176-
within Phacelia phylogenies using a maximum accession) included 89 taxa (42% genus) and the
likelihood approach (Stebbins and Major 1965; expandedplastid partition (126-accession) includ-
Raven and Axelrod 1978; Wood et al. 2009; ed 90taxa(43% genus). Samplingwithin Phacelia
Hallinan and Lindberg 2011a). for the expanded nuclear (176-accession) and
plastid (126-accession) partitions included repre-
Materials and Methods sentatives from all subgenera and sections; the
reduced (61-accession) partition lacked a repre-
Chromosome Numbers in Phacelia sentative from Phacelia sect. Pachyphyllae Wal-
den & R. Patt. Lor the nuclear partition 48 taxa
We reviewed the literature for published were sampled from California, 44 taxa sampled
chromosome counts for taxa in Phacelia and from western North America, 5 taxa were
outgroups sampled in this study (Table 1). Taxa sampled from Central America, and one taxon
are presented alphabetically for outgroups {Eu- was sampled from South America. For the
ploca Nutt., Eriodictyon Benth., Draperia Torr,, plastid partition 53 taxa were sampled from
Hesperochiron S. Watson, Howellanthus [Con- California, 37 taxa were sampled from western
stance] Walden & R. Patt., Nama L., Romanzof- North America, five taxa were sampled from
fia, Tricardia Torr. ex S. Watson) and ingroup Central America, and one taxon was sampled
taxa within Phacelia. Original names for pub- from South America. Accessions ofRomanzoffia
lished counts are noted for synonyms or where were included to assess the relationship to and
different from the current accepted name or monophyly of Phacelia. We included accessions
specimen determination. Chromosome numbers from Hydrophylloideae to briefly assess relation-
for haploid {n) or diploid {2n) counts are given as shipsbetweenPhaceliaand exemplartaxaandfor
published. We chose not to include references purposes of chromosomal evolution. Euploca
reporting unpublished counts or citing personal (Heliotropioideae: Boraginaceae) was included
communications for these analyses. We note ifa as the diploid outgroup to root the tree.
voucher specimen was not cited in the notes Lield collections of fresh plant material were.,
column of the table. No attempt was made to preserved in silica gel for molecular work and
locate and examine all voucher specimens cited voucher specimens were deposited in the Harry
for each published count for this study. Refer- D. Thiers Herbarium at San Lrancisco State
ences may include one or more counts for an University (SLSU). Additional material was
individual taxon, we do not include summary sequenced from banked molecular vouchers with
numbers of the individual counts for each herbarium vouchers received from the William L.
i ( ! < 1 i
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2014] WALDEN ET AL.: PHYLOGENIES AND CHROMOSOME EVOLUTION OF PHACELIA 25
to
17:259-263.
Contributions
Botany
to SIDA:
Contributions
Asteraceae.
mostly
SIDA:
species,
Asteraceae.
Mexican
mostly
and
species,
USA.
Mexican
Miscellaneous
and
3.
USA.
1993:
Miscellaneous
numbers
2.
1996:
chromosome
numbers
Documented
chromosome
1993.
Documented Turner.
15:649-653.
L.
1996. B.
AND
Z.
Botany
Zhao,