Molecular Phylogenetics of Garrya (Garryaceae)

2011·5.4 MB·

by  Dylan O Burge

1
Madrono, Vol. 58, No. 4, pp. 249-255, 2011
MOLECULAR PHYLOGENETICS OF GARRYA (GARRYACEAE)
Dylan O. Burge'
Duke University Department of Biology, Box 90338, Durham, NC 27708
dyIan o burge@gmail com
. . .
Abstract
Garrya (Garryaceae) comprises 15 species of shrubs and small trees restricted to the Americas.
Garrya is taxonomically divided into two subgenera, Garrya and Fadyenia, which differ in
morphology, secondary chemistry, and geographic distribution. The present work uses nuclear
DNA
ribosomal (ITS) sequence data from 11 Garrya species to elucidate phylogenetic relationships
within the genus and test the monophyly of the subgenera. Results strongly support subgenus
Fadyenia as monophyletic, while monophyly of subgenus Garrya is supported only by maximum
parsimony analyses. ITS data do not provide evidence for genetic admixture between the two
subgenera of Garrya in spite ofbroad geographic overlap.
Key Words: Aucuba, California, Eucommia, Fadyenia, Garrya, Garryaceae, ITS, Mexico.
Garrya Douglas ex Lindl. contains 15 species this to the taxonomy, distribution, and biology of
endemic to NorthAmerica, Central America, and the species. Specifically, I test the hypothesis that
the Caribbean (Dahling 1978; Nesom unpub- the Garrya subgenera are monophyletic, with
lished; Table 1; Fig. 1). Garrya species are separate histories of diversification in different
dioecious shrubs and small trees with decussate, geographic regions ofthe Americas.
evergreen leaves and pendulous, catkin-like
inflorescences. The plants are probably wind Materials and Methods
pollinated (Hallock 1930; Dahling 1978; Liston
2003). Garrya is found in a diversity of habitats Genetic Sampling
over its broad geographic range, from cloud
forest to maritime chaparral, but is typically a Sampling of Garrya populations was designed
component of shrublands (e.g., chaparral) or to represent the geographic range of the genus
forests (Dahling 1978). with an emphasis on California, the southwestern
Molecular phylogenetic studies consistently U.S., and Mexico (Table 3; Appendix 1; Fig. 1).
resolve Garrya as sister to the east Asian shrub One sample of Aucuba was obDtaNinAed from a
genus Aucuba (Soltis et al. 2000; Bremer et al. garden planting in Cahfornia. from 22
2002), which together comprise Garryaceae Garrya individuals was studied, representing 1
(APG 2003). These results confirm a close of the 15 species currently recognized (Dahling
relationship that has long been hypothesized on 1978; Nesom unpublished). For the species
the basis ofmorphology and chemistry (reviewed occurring in the U.S., voucher specimens were
identified according to Nesom (unpublished); for
in Liston 2003). Molecular phylogenetic studies
also support a close relationship between Gar- all exclusively Latin American taxa, identifica-
ryaceae and the monotypic east Asian tree tions were according to DahDliUnKgE(1978). Voucher
Eucommia (Eucommiaceae), which togethercom- specimens are deposited at (Appendix 1).
prise the euasterid order Garryales (APG 2003).
Garrya is divided into two subgenera, Garrya Molecular Methods
(6 spp.) and Fadyenia (9 spp.), which differ in Genomic DNA was extracted from silica-dried
geographic distribution, inflorescence morpholo- leaf tissue using the DNeasy Plant Mini Kit
gy, and secondary chemistry (Dahling 1978; (Qiagen, Germantown, MD) according to the
Table 2). Subgenus Fadyenia has its center of manufacturer's instructions. Polymerase chain
diversity in Mexico, while subgenus Garrya reactions were performed using Qiagen Taq
reaches peak diversity in the western U.S. The DNA Polymerase. Amplification was performed
geographicdistribution ofthe subgenera overlaps usingan initial incubation at 94 C for 10 min and
in the southwestern U.S. and Mexico (Dahling 30cycles ofthree-step PCR (1 min at 94 C, 30 sec
1978). Research presented here aims to elucidate at 55°C, and 2 min at 72°C), followed by final
phylogenetic relationships in Garrya and relate extension at 72'C for 7 min. I amplified the
nuclear ribosomal ITS region (ITS 1, 5.8S, and
'Present address: National Herbarium ofNew South ITS2) using the primers ITS4 (White et al. 1990)
Wales, Royal Botanic Gardens, Sydney, Mrs Mac- and ITSA (Blattner 1999). I amplified the /r/7L-F
quaries Road, New South Wales, 2011, Australia. plastid region, comprisingthe trn\^ intron and the
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250 [Vol. 58
Table 1. Garrya Speciesand Sampling. Sampling: number ofpopulations sampled for phylogenetic analysis
(Table 3); Distribution: geographic distribution of the species (SW U.S.: southwestern United States); CFP:
indicates whether species occurs in the California Floristic Province; Flower: range ofknown flowering times for
species (Dahling 1978). ^ subspecies are recognized by Dahling (1978) and/or Nesom (unpublished): G. ovata: 3
subspecies; G. laurifolicr. 4.
Species Sampling Distribution CFP Flower
Subgenus Fcidy^nici
G.fadyena Hook. u Greater Antilles Dec-Feb
G. glaberrima Wangerin 3-> E Mexico Mar-May
G. grisea Wiggins 11 Mexico (Baja California) A Feb-Apr
G. laurifolia Benth.^^ Mexico and Central America Dec-Apr
G. liuaheimeri 1orr. 11 SCMW/ TUT.SC., AM/eT^x,i,;c^o^ Mar-May
G. lougifolia Rose 0 Mexico Jan-Mar
G. ovata Benth." 11 SW U.S. and Mexico Mar-Apr
G. salicifolia Eastw. u Mexico (Baja California Sur) Aug-Dec
G. wrightii Torr. 3 SW U.S. and Mexico Apr-Aug
Subgenus Garrya
G. hux(folia A. Gray 1 U.S. (CA and OR) X Feb-Apr
&
G. corvoriuri Standi.
Steyerm. 0 Guatemala Dec-Jan
G. elliptica Douglas ex
Lindl. 2 U.S. (CA and OR) X Dec-Feb
G. flavescens S. Watson 3 U.S. (AZ, CA, NV NM, UT) and X Feb-Apr
Mexico
G.fremontii Torr. 3 U.S. (CA, OR, WA) X Jan-Apr
G. veatchii Kellogg 2 U.S. (CA)and Mexico (Baja California) X Jan-May
trnh-¥ intergenic spacer, using primers c and fof Because ofthis low level ofvariation, rr«L-F was
Taberlet et al. (1991). Excess primer and dNTPs not sequenced in additional plants, and was
wBieorleabrse,moIvpsewdicuhs,inMg Aexon[uNcElBe]a;se0.I2(uNneitws/|EanlgPlaGnRd aalbiagnndmoenntedandintrfeeavbourildoifng.ITS for subsequent
product) and antarctic phosphatase (NEB; 1.0 The 23 new ITS sequences (22 Garrya and 1
unit/|al PGR product) incubated for 15 min at Aucuba) were supplemented with an ITS se-
37'G followed by 15 min at 80 G. For sequenc- quence for Eucommia ulmoides Oliv. from Gen-
ing. Big Dye chemistry (AppHed Biosystems, Bank (Table 3). All DNA sequences were assem-
Foster Gity, GA) was utilized according to the bled and edited using Sequencher 4.1 (Gene
manufacturer's instructions. Sequences were de- Codes Corporation). Edited sequences were
termined on an Applied Biosystems 3100 Genetic deposited in GenBank (/r«L-F: JN234721-32;
Analyzer at the Duke University Institute for ITS: Table 3). ITS sequences were aligned using
Genome Science and Policy Sequencing and MUSCLE (Edgar 2004) under default settings.
Genetic Analysis Facility. Due to ambiguity, a 22 bp region of ITSl was
excluded from all subsequent analyses. The
Sequences and Alignment alignment was deposited in TreeBase (Study
11755).
A total of23 ITS and 12 /r/7L-F sequenceswere
A
generated for the present study. preliminary Phylogenetic Analysis
alignment of trnl.-¥ revealed that the nine
sequenced Garrya species shared a nearly identi- Trees were reconstructed using Bayesian,
cal sequence; there was just a single nucleotide maximum likelihood (ML), and maximum parsi-
substitution difference among the species, a mony (MP) techniques. Trees were rooted using
change unique to G. grisea Wiggins (D.O. Burge Eucommia ulmoides (APG 2003). Bayesian phy-
778; Table 3). In addition, two insertion/deletion logenetic analyses were conducted using the best-
events were present within Garrya, 1) a 1 bp fit model of evolution from AIG output of the
length diffei"ence within the trn¥ intron, where a program MrModeltest (GTR + G; Nylander
poly-T region was one bp longer in two 2004). Sampling of trees was performed using
accessions of G. elliptica Douglas ex Lindl. the program MrBayes 3.0 (Ronquist and Huel-
(D.O. Burge 382 & 386; Table 3) than in sMeGnbMecGk 2003). Three separate runs of 1 X 10^
remaining Garrya, and 2) a 2 bp length difference generations were performed using one
in the /rnL-F intergenic spacer, where a poly-T heated and three cold chains, sampling every
region wastwo bp longerin members ofsubgenus 1000 generations. Independent chains were in-
Fadyenia relativetomembersofsubgenus Garrya. spected for convergence (standard deviation of
1
2011] BURGE: PHYLOGENETICS OF GARRYA 251
1160
rwv. I
^
148
382
378yV37'
H9,
1036'""^
778
)750
1041
11252, t25<
^1239,
O
;:^y^^^6, i2if
12211QX 1225
Legend
O Collections
GarryaDistribution
- 200km
Fig. 1. Garrya distribution and sampling. Distribution of Garrya indicated by dark gray shading (data from
participants of the Consortium of California Herbaria, 2011). Sampling locations indicated by white circles
(Table 3).
split frequencies nearing 0.001). Log-likelihood lengths and posterior probabilities (PP) of
for the sampled tree was plotted and examined in clades. Consensus phylograms were built for
Microsoft Excel to assess convergence and each of the three independent runs using
determine an appropriate burn-in period (Ron- MrBayes (Ronquist and Huelsenbeck 2003).
quist and Huelsenbeck 2003). A total of 1 X 10' Following inspection to verify similarity of the
generations (100 trees) were eliminated as burn- results, trees from all three runs were combined
in, leaving 9 X lO'^ generations (950 trees) in a consensus phylogram. Maximum likelihood
of explored tree space for computing branch tree building was performed in GARLI v 1.0
Table 2. Morphologicaland Distributional Comparison Between the Two Subgenera of Garrya.
Description
Character Subgenus Garrya Subgenus Fadyeuia
9 inflorescence Compact, pendulous, unbranched Loose, erect, branched
Ovary appendage Small, epigynous Large, foliaceous, partially adnate
Paired floral bracts
Flowers per pair Three One
Fusion ofpair Basally connate, forming a cup Distinct to partially adnate basally
Size in 9 Reduced in size, not leaf-like At least proximal large and leaf-like
Geographic distribution Western U.S. and northern Mexico Western U.S., Mexico, Central
America, and Caribbean
S
.
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252 [Vol. 58
Table 3. Collection Number and Provenance for Voucher Specimen (Appendix 1) and GenBank
Accession Numbers for ITS Sequences. All vouchers deposited at DUKE.
Taxon Collection number and provenance GenBank 11
Aucubajaponica Thunb. DO. Burge 363, Butte Co., CA JN234733
Eucommia idmoides A. Gray AY650006
Garrya, subgenus Fadyenia
G. glaberrima Wangerin D.O. Burge 1025, Hidalgo, Mexico JN234743
D.O. JJLII^C 1 1 i^LlCVl„? J_/CiJll, iVlCAlCLJ JN234744
DO. Burge 1225, Tamaulipas, Mexico JN234745
G. grisea Wiggins DO. Burge 778, Baja California, Mexico JN234746
G. iQuviJolici Benth. DO. Burge 1218, Nuevo Leon, Mexico Ji>z.J^1'\1
DO. Burge 1252, Chihuahua, Mexico TN234748
\yJj,. iIJI\ril.tlllfLilcIlr^HCeiiflIil.lyJlV, DDOO.. BBuurrggee 715202,1,TrNauveivsoCoL.e,onT,XMexico
\j, WTvfiitbyrlitlii1i1 '1~Vc\T1V DDOO.. BBuurrggee 913243,9,PDiumraanCog.o,,AMZexico TrNN723344775534
DO. Burge 1253, Chihuahua, Mexico TN734755
Garrya, subgenus Garrya
G. buxifoUa A. Gray D.(J. Burge lloO, Josephine Co., OK JN234734
G. elliptica Douglas ex Lindl. DO. Burge 382, Monterey Co., CA JN234735
DO. Burge 386, Marin Co., CA JN234736
G.flavescens S. Watson DO. Burge 370, Kern Co., CA JN234737
DO. Burge 419, Yavapai Co., AZ JN234738
DO. Burge 1036, Baja California, Mexico JN234739
G.fremontiiTorr. DO. Burge 353, Butte Co., CA JN234740
DO. Burge 362, Humboldt Co., CA JN234741
D.O. Burge 1148, Tuolumne Co., CA JN234742
G. veatchii Kellogg DO. Burge 378, San Luis Obispo Co., CA JN234751
D.O. Burge 1041, Baja California, Mexico JN234752
(Zwickl 2006). Two search replicates of 1 X 10^ characters, 22 of which were excluded (see
generations were performed in a single execu- above). Ofthe 674 included characters, 189 were
tion with a random starting tree. Other param- variable and 48 were parsimony informative.
eters were kept at default values. Statistical
support was inferred with 100 replicates of Phylogeny
bootstrap reweighting (Felsenstein 1985) using 5
X 10"^ generations per replicate. The majority Bayesian, ML, and MP analyses provided
rule consensus tree was calculated using the 100 similar topologies and levels of support (Fig. 2;
best bootstrap trees. Maximum parsimony TreeBase Study 11755). Maximum parsimony
phylogenetic analysis was carried out using analysis resulted in 147 equally parsimonious
PAUP* v 4.0 (Swofford 2000). Heuristic search- trees (length = 217, CI = 0.97, RI = 0.97). A
es used 1000 random sequence addition repli- total of eight nodes are found in the strict
cates and tree bisection-reconnection branch consensus ofthese trees (Fig. 2). Overall, Garrya
swapping. Nonparametric bootstrap analysis is strongly monophyletic (Bayesian PP 0.99; MP
(Felsenstein 1985) was conducted using 100 bootstrap 100%; ML boostrap 95%; Fig. 2);
prsaenuddoormepsleiqcuaetnecseaandddihteiuornisrteipclicsaettetsi.ngsInwailtlhM1P0 msounbogpehnyulseFtaidcye(nBiaayeissiaalnsoPPstr1o.n0g;lyMsPuppbooorttsetdraaps
analyses, gaps introduced by the alignment 100%; ML boostrap 100%), with several moder-
process were treated as missing data. ately-supported groupings within it. Though
subgenus Garrya is monophyletic in the strict
MP
Results consensus cladogram from analysis, and
receives 89% MP bootstrap support, this group
DNA Sequences is not supported in Bayesian or ML analyses. In
addition, a grouping ofGarrya ovata Benth. with
The ITS region for A. japonica Thunb. was Garrya lindheimeri Torr. is strongly supported
605 bp in length. In Garrya this region varied (Bayesian PP 1.0; MP bootstrap 86%; ML
from 622 to 624 bp; in all members of subgenus bootstrap 84%), as in a clade containing all
Garrya the region was 624 bp long while in sampled populations of Garrya glaberrima Wan-
Dsu.bOg.enBuusrgFeady77e8n;iaTaitblvaeri3e)dtforo6m2362b2p.(GT.hgerisIeTaS, gPePrin0.a9n9;d oMnPe Gabroortysatrlaauprif9o3li%a;BeMntLh. b(oBoatysetsriaapn
alignment (TreeBase Study 1755) contained 696 90%). However, none ofthe seven Garrya species
1
2011] BURGE: PHYLOGENETICS OF GARRYA 253
#
- G.laurifolia(^252)
#
- G.wrightii0239)
<50/58 #
- G.wrightii{^253)
0.92 o
55/67 - G.grisea(778)
0
0.93 - # G.wrightii(934)
86/84 - G.lindheimeri(750)
100/100 #
1.00 - G.ovata(1221)
#
1.00 - G.glabenrima(1225)
62/61 #
- G.laurifolia{^2^8)
93/90 0.87 •
- G.glaberhma(1216)
0.99 #
- G.glaberhma(1025)
o
- G.veatchii(^04^)
o
- G.veatchii(378)
o
- G.elliptica(382)
o
- G.fremontii(353)
o
- G.e///pf/ca(386)
89/<50 o
- G.flavescens(1036)
<0.50 o
- G./jux/fo//a(1160)
o
- G.fremonrt;(1148)
o
- G.flavescens(370)
•
- G.flavescens(419)
o
- G.fremontii(362)
- £.ulmoides
- A.japonica
Fig. 2. Strict consensus of 147 equally parsimonious trees recovered in maximum parsimony (MP) phylogenetic
MP
analysis, with support values from 100 bootstrap replicates above branches, at left. Tree is rooted using E.
ulmoides. Support from maximum likelihood bootstrap (above branches, at right) and Bayesian analysis (below
branches) mapped on tree. Species names followed by D.O. Burge collection numbers. Open circles indicate
collections obtained fromwithin theCalifornia Floristic Province (CFP); darkcircles are fromoutsideoftheCFP.
represented by more than one sampled plant are morphologically distinct in the parts ofnorthern
recovered as monophyletic (Fig. 2). Mexico where they occur sympatrically, though
hybrids may occasionally form (Nesom unpub-
Discussion lished).
It is also noteworthy that no individual Garrya
Phylogenetic Relationships species is monophyletic (Fig. 2). One potential
exception is G. glaberrima; the three included
ITS trees strongly support subgenus Fadyenia, individuals of this species group together rela-
as circumscribed by Dahling (1978; Fig. 2). The tively strongly with a single individual of G.
Phylogenetic isolation of subgenus Fadyenia is laurifolia (Fig. 2). The strong divergence of G.
supported by morphology, secondary chemistry glaberrima from remaining members ofsubgenus
(Dahling 1978), and geographic distribution Fadyenia is supported by the unusual morphol-
(Table 2). By contrast, the monophyly of subge- ogy and phytochemistry of the species (Dahling
nus Garrya is strongly supported only by 1978); presence of one individual of G. laurifolia
maximum parsimony trees (Fig. 2). The lack of (D.O. Burge 1281) in this group might be
support for subgenus Garrya that is seen in explained by geneflow, as this individual was
maximum likelihood and Bayesian analyses may collected in an area where G. glaberrima occurs
represent an artifact of analysis due to the small (D.O. Burge 1216; Table 3; Figs. 1 and 2). The
size of the ITS dataset. Future studies should overall lack of monophyly for individual species
utilize additional genes from both the chloroplast of Garrya is noteworthy as it is consistent with
and nuclear genomes. the action of incomplete lineage sorting (Maddi-
The strong relationship between G. ovata and son and Knowles 2006) due to shallow genetic
G. lindheimeri indicated by ITS is supported by divergence among species, possibly exacerbated
the similar morphology of these species (Nesom by geneflow. Hybrids are not frequently observed
unpublished). Indeed, the similarities are so great in the wild (Dahling 1978; D.O. Burge, personal
that G. lindheimeri was treated as part of G. observation; but see Munz and Keck 1968), and
ovata, at the subspecies rank, by Dahling (1978). the extent of geneflow among species of Garrya
Nevertheless, the species are ecologically distinct has never been directly studied. Thus, incomplete
over most oftheir geographic range, and remain lineage sorting stands as the most probable
MADRONO
254 [Vol. 58
explanation for the general lack of phylogenetic material using recombinant PCR. Biotechniques
cohesion among populations of individual spe- 27:1180-1185.
cies. Nevertheless, the present study does not Bremer, B., K. Bremer, N. Heidari, P. Erixson,
include all species, and is based on a small sample LRE.RSGJ.O,OalnmsdteE.adB,arak.hoar.daArnidaenr.be2r0g0,2.MP.hylKoagle--
ofpopulations; analysis ofadditional species and neticsofasteridsbasedon3codingand3non-coding
populations might reveal greater phylogenetic chloroplast DNA markers and the utility of non-
affinity among populations ofindividual species. coding DNA at higher taxonomic levels. Molecular
In addition, the present study is based on a very Phylogenetics and Evolution 24:274—301.
small sample of DNA sequence data; additional Dahling, G. V. 1978. Systematics and evolution of
data, ideally from both the chloroplast and Garrya. Contributions to the Gray Herbarium of
nuclear genomes, might provide greater phyloge- Harvard University 209:1-104.
Edgar, R. C. 2004. MUSCLE: multiple sequence
netic support for individual species.
alignmentwithhigh accuracyandhighthroughput.
Nucleic Acids Research 32:1792-1797.
Diversification of Garrya in the Americas Felsenstein,J. 1985. Phylogeniesandthecomparative
method. American Naturalist 125:1-15.
SubgenusFadyeniarepresentsalineagethathas Hallock, F. a. 1930. The relationship ofGarrya. The
diversified in the mountainous regions of the development of the flowers and seeds of Garrya
southwestern U.S., Mexico, CentralAmerica, and and its bearing on the phylogenetic position ofthe
genus. Annals ofBotany 176:771-812.
the Greater Antilles (Fig. 1). If subgenus Garrya LiSTON, A. 2003. A new interpretation of floral
is monophyletic, as suggested by some phyloge- morphology in Garrva (Garryaceae). Taxon
netic analyses (Fig. 2), the group would represent 52:271-276.
a diversification that is focused in the CaUfornia Maddison, W. p. and L. L. Knowles. 2006. Inferring
Floristic Province (CFP) of western North phylogeny despite incomplete lineage sorting.
gAemoegrriacpahi(cTaobvleerl1a,pFiogf.t2h)e.seIntwspoitgeroofuptsheinwitdhee MuNZSy,stPe.maAt.icABNiDoloDg.y 5D5.:2K1ec30k.. 1968. A California
southwestern U.S. and Mexico, which should flora and supplement. University of California
Press, Berkeley, CA.
pprheysleongtenoeptpiocrtruensiutlitsesdfoorniontteprrborveieddienge,vimdoelnecceulfaorr Nyladinsdterirb,utJe.dAb.yA.th2e00a4u.thMorrM.odEevlotleustti,onva2r.yPBriooglroagmy
geneflowbetween the two subgenera ofGarrya. It Centre, Uppsala University, Uppsala, Sweden.
is possible that this lack ofgeneflow is driven by Ronquist, F. andJ. P. Huelsenbeck. 2003. MrBayes
differences in flowering time, as indicated by a 3: Bayesian phylogenetic inference under mixed
slight tendency toward earlier flowering in subge- models. Bioinformatics 19:1572-1574.
nus Garrya as compared to subgenus Fadyenia SoLTis, D. E., P. S. SoLTis, M. W. Chase, M. E.
tl(oiTcoaanbtlieoofn1s).siTtnhaigtsgheeirdesedoautifshlwsoeuwspetpreoirrnntgeUd.tSib.myewthhaeetroebssemevreevrmaa--l WNSM.i.oxMRHo.T.n,,SHDwa.EahNnnCS.d,EANSl,.J.bBLa..Sc.HhMo,.FoatMPr,.rriiMZcsa.e.n,iFW.s2,.0F0aV0J.y..,SKrAaMenv.sogsAli,xaotisKenp.leelnrC,,m.
bers ofeach subgenus occur as part of the same phylogeny inferred from 18S rDNA, r/^cL, and
plant communities (D.O. Burge, personal obser- atp^ sequences. Botanical Journal of the Linnean
vation). Society 133:381-461.
Swofford, D. L. 2000. PAUP*. Phylogenetic analysis
Acknowledgments using parsimony (*and other methods). Version
4.01b10, Sinauer Associates, Sunderland, MA.
The author thanks the Duke University Systematics Taberlet, p., G. Ludovic, G. Pautou, and J.
Discussion Group for constructive criticism of this Bouvet. 1991. Universal primers for ampHfication
work. The author also thanks Katherine Zhukovsky of three non-coding regions of chloroplast DNA.
and Kaila Mugford for assistance with lab work. Plant Molecular Biology 17:1105-1109.
Funding was provided by the Duke University Depart- White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990.
ment of Biology. Amplifications and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. Pp. 315-
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—
2011] BURGE: PHYLOGENETICS OF GARRYA 255
Appendix 1 Creek), Butte Co., CA; D.O. Burge 362, Trinity River
canyon. Poison Gulch, Humboldt Co., CA; D.O. Burge
Sampled Individualsof Garrya and Aucuba 1148, North Fork Tuolumne River watershed. Bald
Mount—ain, Tuolumne Co., CA. G. ^lahen-ima Wan-
Collector and number followed by description of gerin D.O. Burge 1025, Cerro Juarez, near summit,
locality. All specimensdeposited intheDukeUniversity Hidalgo, Mexico; D.O. Burge 1216, Cerro El Potosi,
Herbarium (DUKE). eastern slope, Nuevo Leon, Mexico; D.O. Burge 1225,
—
Aitcuha japonica Thunb. D.O. Burge 363, City of Sierra E—l Pedregoso, Tamaulipas, Mexico. G. grisea
Chico, 1469 H—umboldt Rd, Butte Co., CA. Gavvya Wiggins D.O. Burge 778, Sierra San Pedro —Martir,
buxifolia Gray D.O. Burge 1160, Swede Creek water- Baja California, Mexico. G. laurifolia Benth. D.O.
shed, roadside on FR 2524 (road to Spalding Mill), Burge 1218,CerroElPotosi,easternslope,NuevoLeon,
Josephine Co., OR. G. elliptica Douglas ex Lindl. Mexico; D.O. Burge 1252, Cascada de Bas—aseachi area.
D.O. Burge 382, Seaside, eastern terminus of Kimball Chihuahua, Mexico. G. lindheimeri Torr. D.O. Burge
Avenue near Fort Ord Military Reservation, Monterey 750,CityofAustin, Mayfield ParkandNaturePreserve,
Co., CA; D.O. Burge 386, Mount Tamalpais, roadside Travis Co., TX. G. ovata Benth.—D.O. Burge 1221,
onEast Ridgecrest Boulevard,nearMiddlePeak, Marin Sierra L—os Soldados, Nuevo Leon, Mexico. G. veatchii
Co., CA. G. flavescens S. Watson D.O. Burge 370, Kellogg D.O. Burge 378, Cuesta Ridge, San Luis
Ball Mountain, western slope, along Caliente Bodfish ObispoCo., CA; D.O. Burge 1041, Isla Cedros, N slope
Rd, Kern Co., CA; D.O. Burge 419, Wilson Mountain, ofCer—ro Redondo, Baja California, Mexico. G". wn'ghtii
Wilson Mountain Trail, Yavapai Co., AZ; D.O. Burge Torr. D.O. Burge 934, Santa Catalina Mountains,
1036, Cerro Bola, easte—rn slope, Baja California, Pima Co., AZ; D.O. Burge 1239, Sierra de Coneto,
Mexico. G.frenwntiiTon D.O. Burge 353, Doe Mill western slope, Durango, Mexico; D.O. Burge 1253,
.
Ridge (ridge between Butte Creek and Little Chico Cascada de Basaseachi area. Chihuahua, Mexico.