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Zoological Journal of the Linnean Society, 2015. With 5 figures
Geometric morphometric and phylogenetic analyses of
Arizona Sky Island populations of Scaphinotus petersi
Roeschke (Coleoptera: Carabidae)
KAREN A. OBER* and CRAIG T. CONNOLLY
Department of Biology, College of the Holy Cross, 1 College St., Worcester, MA 01610, USA
Received 20 August 2014; revised 23 December 2014; accepted for publication 25 February 2015
Scaphinotus petersi Roeschke, 1907 (Carabidae) is a ground beetle endemic to Sky Islands in south-eastern Arizona.
Previous taxonomic studies described several subspecies with morphological differences inhabiting geographically
isolated mountain ranges. We combined molecular sequence data and morphometric data, especially head and pronotum
shape analyses, to examine the variation and divergence in subspecies and isolated montane populations. In this
study, we employ a combination of distance morphometrics as well as geometric morphometrics to quantify the
level of morphological variation, and to test the hypothesis that geographically distinct populations of S. petersi
are phenotypically distinct. Results suggest that these isolated populations have diverged morphologically and genetically. Phylogenetic analyses identified two monophyletic lineages within the species that correspond generally
to pronotum shape. We observed significant morphological variation among most montane populations in of S. petersi,
with the pronotum shape as the clearest delimiting trait.
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
doi: 10.1111/zoj.12269
ADDITIONAL KEYWORDS: Arizona – beetles – geometric morphometrics – Madrean Sky Islands –
molecular systematics.
INTRODUCTION
Studying how variation in geography and ecological
interactions affects patterns of morphological divergence can lead to a better understanding of evolutionary processes that generate new species. Morphological
divergence may be caused by chance events such as
genetic drift or founder effects, or may be the result
of natural selection arising from geographically patterned variation in ecological factors. Studying geographic variation within species when evolutionary
divergence may be in progress provides an opportunity to observe the speciation process in action, and
may reveal both recent and historic changes in geology,
climate, ecology, or dispersal patterns contributing to
population subdivision.
Geographic isolation is a major cause of the evolution of new taxa (Coyne & Orr, 2004). Geographically
*Corresponding author. E-mail: [email protected]
isolated mesic refuges, such as those found in the southwest mountains of the USA, have been important areas
of diversification for carabid beetles during periods of
dry climate (Noonan, 1992). The Sky Islands (Heald,
1951), also called the Madrean Archipelago, form a
unique complex of mountain ranges and ecosystems,
encompassing nearly 270 000 km2 in south-eastern
Arizona, south-western New Mexico, and northern
Mexico. The mountains form discontinuous chains separated by two major rivers: the Gila River and the San
Pedro River (Fig. 1). At present, hot and dry desert
grasslands and scrub in the valleys between mountain ranges (‘the sea’ between the Sky Islands) act as
a barrier to the movement of upland forest species.
Similar to the way in which saltwater seas isolate biota
on oceanic islands, these valleys separate montane habitats, and therefore limit genetic interchange between
populations, and create environments with high evolutionary potential. The Sky Island ecosystems, renowned for their biodiversity (Lomolino, Brown & Davis,
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
1
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K. A. OBER AND C. T. CONNOLLY
Figure 1. A, study location; distribution area of Scaphinotus petersi is circled. Habitats above 1830 m a.s.l. are shown
in black, and habitats between 1500 and 1830 m a.s.l. are shown in grey. B, shaded relief map of study area. Black dots
denote the sampling localities of S. petersi used in this study (see Table 1), abbreviated as follows: C, Chiricahua Mountains; H, Huachuca Mountains; P, Pinal Mountains; PN, Pinaleño Mountains; R, Rincon Mountains; SA, Sierra Ancha
Mountains; SC, Santa Catalina Mountains; SR, Santa Rita Mountains; WM, White Mountains. Figure modified from
Ober et al. (2011).
1989), are considered a biodiversity ‘hot spot’ (Spector,
2002) for their endemic species richness, including many
threatened and endangered species. In combination,
the geographical and ecological uniqueness of the Sky
Islands create a natural laboratory to examine morphological divergence resulting from the evolutionary
dynamics of vicariance. Ecosystems atop these mountain ranges are sufficiently separated by the dry, hot
lowland habitats, such that their remoteness promotes the differentiation of organisms that live there.
Geographically isolated mesic refuges, such as those
in the south-west mountains of the USA, have been
important areas of diversification for carabid beetles
during periods of dry climate (Noonan, 1992). Carabidae
(ground beetle family) is a large family of insects, containing approximately 40 000 described species (Lorenz,
2005). The snail-eating beetles of the genus Scaphinotus
belong to the carabid tribe Cychrini. Cychrines consist
of about 150 species in four genera, and are restricted to the Northern Hemisphere; the Cychrini genus
Scaphinotus, found only in North America, radiated
about 35 Mya (Scudder, 1900; Osawa, Su & Imura, 2004)
into 55 species (Lorenz, 2005). Scaphinotus petersi
Roeschke, 1907, a large ground beetle, is endemic, confined exclusively to moist coniferous forests in southeastern Arizona at elevations > 1800 m a.s.l. Scaphinotus
petersi is a specialist predator of land snails; it uses
elongated and narrow mouthparts to penetrate and
extract the soft parts of terrestrial snails (LaRochelle,
1972; Digweed, 1993). Scaphinotus petersi, like other
Scaphinotus species, is flightless, with reduced or absent
flight wings under fused elytra. At present, six subspecies of S. petersi have been characterized by Ball
(1966) based on isolated geographic locations and some
morphological differences, including colour variation
of the dorsal surface and type of punctuation of the
pronotum. Ball (1966) also found that S. petersi beetle
populations on several Sky Island mountain ranges have
some distinct differences in size, and some differences in pronotum and leg characteristics, albeit that
these are not clear-cut. It appears there are visual morphological differences between S. petersi from different mountain range populations. In addition, recent
molecular phylogeographic studies indicate structured genetic variation in mitochondrial DNA with some
deep genetic divergences among montane beetle populations (Ober et al., 2011; Mitchell & Ober, 2013). All
six S. petersi subspecies live exclusively on mountains in the sub-Mogollon Sky Island area of Arizona
(Fig. 1; Table 1).
Here we examine patterns of variation among isolated populations of S. petersi by inferring evolutionary relationships among populations using molecular
sequence data, and by quantifying morphological variation with geometric morphometrics to evaluate geographical patterns of differentiation. In this paper we
examine variation in size, but we also included additional morphological characters that seem to have discriminatory value, and use new methods to examine
morphological divergence. Principally, for the first time
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
MORPHOMETRICS OF SCAPHINOTUS PETERSI
Table 1. Scaphinotus petersi subspecies, mountain range
collection locality, and number of specimens included in the
morphological study
Subspecies
Mountain
range
No.
females
No.
males
catalinae
corvus
biedermani
biedermani
petersi
petersi
grahami
grahami
kathleenae
Catalina
Chiricahua
Huachuca
Rincon
Pinal
Sierra Ancha
Pinaleño
White
Santa Rita
30
24
7
3
7
0
33
5
3
55
21
7
3
6
2
27
4
5
Total
85
45
14
6
13
2
60
9
8
242
in this species, we investigate how morphological shapes
vary in different populations through geometric
morphometrics. Geometric morphometrics have clear
advantages over linear measurements and their ratios,
which cannot capture intricate changes in the shape
of a particular feature, can be correlated with size, and
can underestimate geometric complexity. We address
the following questions: (1) is there morphological shape
variation, and also size variation, in the species
S. petersi; (2) have isolated populations of S. petersi
evolved different morphological shape and size characteristics; and (3) do morphological differences follow
a phylogeographic pattern?
MATERIAL AND METHODS
DNA
EXTRACTION, AMPLIFICATION, AND SEQUENCING
A total of 70 S. petersi samples from eight mountain
ranges (Fig. 1; Table S1) were included in the
phylogenetic analysis along with Sphaeroderus lecontei
Dejean, 1826, which served as an out-group. Specimens from all subspecies and populations in Table 1
were included in the molecular analyses, except for the
Sierra Ancha and White Mountain populations. We collected 49 new DNA sequences from S. petersi. Genomic
DNA was extracted following the protocol outlined in
Maddison, Baker & Ober (1999). Polymerase chain reactions (PCR) were performed using a modification of
the procedure described in Maddison et al. (1999). Reactions used an annealing temperature of 53–56 °C.
This procedure was used to amplify a 300-base pair
portion of ND1, a 500-base pair portion of COI, and
a 1000-base pair region of 28S rDNA. Eurofins MWG
Operon carried out DNA sequencing using an Applied
Biosystems ABI 3730 48-capillary DNA analyser with
Big Dye Terminator Technology, according to the manufacturer’s protocols (Applied Biosystems). DNA se-
3
quence data were visualized using SEQUENCHER 3.0
(Gene Codes Corp.). Sequences were easily aligned by
eye using MESQUITE 2.75 (Maddison & Maddison,
2011). Data matrices are available from the corresponding author, K. A. Ober. Coding sequence data plus
an adjacent portion of mitochondrial tRNA (mtRNA)
were available for a total of 68 of the 71 samples for
ND1, and coding sequence data were available for 51
of the 71 samples for COI and for 35 of the 71 samples
for 28S. The sequences generated for this study have
been deposited in GenBank, and their accession numbers
are listed in Table S1.
PHYLOGENETIC
RECONSTRUCTION
Phylogenetic patterns were examined by inferring
phylogenetic relationships from combined ND1 + mtRNA,
COI, and 28S sequence data. The combined data set
(3804 characters) was partitioned in four unlinked
subsets (COI positions 1 + 2, ND1 positions 1 + 2,
COI + ND1 position 3, and mtRNA + 28S). Maximumlikelihood searches were completed using GARLI 2.0
(Zwickl, 2011) using an HKY + I + G model of evolution for each subset. The other search settings were
set to default settings. The searches employed a heuristic search strategy and were repeated 1000 times,
starting from random trees and keeping only the tree
with the best likelihood score. Support for the relationships found in these searches was evaluated by 200
replicate bootstrap analyses with 100 addition sequences per replicate. Bayesian analyses were completed in MRBAYES 3.2 (Ronquist & Huelsenbeck, 2003)
using four runs of 100 million generations each. The
same partition strategy and model of evolution as above
was used. Each run used four separate chains, sampling every 1000 generations. Independent runs were
combined using LOGCOMBINER 1.5.4 (Rambaut &
Drummond, 2010). For each analysis, the trees in a
burn-in period were excluded (the first 25% of the runs)
and the majority-rule consensus tree of the remaining trees was calculated by PAUP* (Swofford, 2002),
to determine the Bayesian posterior probabilities of the
clades. The average standard deviation of split frequencies was well below 0.01 and all parameters appeared to have reached stationarity.
MORPHOMETRIC ANALYSES
A total of 112 female and 130 male S. petersi specimens were examined from all six subspecies in nine
mountain ranges in south-eastern Arizona (Fig. 1). The
specimens examined belong to the following collections: Carnegie Museum of Natural History (ICCM),
California Academy of Sciences (CAS), UC Berkeley
Essig Museum of Entomology (UCBC), and the corresponding author’s personal collection (KAO). The
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
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K. A. OBER AND C. T. CONNOLLY
Figure 2. A, head shape landmarks on female Scaphinotus petersi biedermani from Rincon Mountains. Pronotum shape
landmarks: (B) Scaphinotus petersi kathleenae male from Santa Rita Mountains; (C) Scaphinotus petersi biedermani female
from Rincon Mountains.
number of specimens studied for each subspecies and
population is indicated in Table 1. For each specimen, we collected digital images using a SPOT Idea
3MP colour digital camera mounted on an Olympus
SZX7 stereoscopic dissecting microscope using SPOT
imaging software (Diagnostic Instruments, Inc.). We
recorded the following measurements: length of the hind
leg (femur + tibia + tarsus); head length – distance from
anterior margin of clypeus to posterior margin of compound eye, usually measured on left side of head; head
width – maximum transverse distance across vertex
and compound eyes; pronotum length – distance from
the anterior margin to the posterior margin, measured along the midline; elytra length – distance from
posterior tip of scutellum to apex of longer elytron; and
total length – sum of head, pronotum, and elytral length,
as in Ball (1966). Males and females were analysed
separately because of sexual dimorphism in this species.
All measurements were log-transformed to normalise
the data (Shapiro–Wilk’s W-test). To evaluate the degree
of measurement error, we performed double-blind repeat
digital imaging and measurements for each trait for
ten specimens. There were no significant differences
between any of the original and repeated measurements (Student’s t-test, P = 0.26–0.78). We used a oneway analysis of variance test (ANOVA) and the Tukey–
Kramer highly significant difference (HSD) test in
JMPv9.3 (SAS Institute, Inc., 2012) to test the null
hypothesis of no differences in leg length, total body
length, head length, and head width among subspecies and populations.
The x- and y-coordinates of ten homologous pronotum
landmarks and six homologous head landmarks (Fig. 2)
were digitized for each specimen using TPSDIG 2.16
(Rohlf, 2006). We used MORPHOJ (Klingenberg, 2011)
to investigate the number and type of differences in
prontum shape and head shape between subspecies and
populations. The raw coordinates of all specimens were
aligned (i.e. translated, rotated, and scaled to match
one another) using the Procrustes generalized orthogonal least-squares (GLS) superimposition method, leaving
only shape variation for further analyses (Rohlf & Slice,
1990). A multivariate analysis of variance test
(MANOVA; SAS Institute & Inc, 2012) was used to test
for significant differences among populations and subspecies in the distance measurements between landmarks along the pronotum and head outline.
Partial warps were calculated using the Procrustes
shape residuals. To survey the patterns of variation
in pronota and heads, within and between subspecies and mountain ranges, we used principal components analyses (PCA) of partial warps. Canonical variate
analyses (CVA) with MANOVA of the partial warp scores
matrix were performed to compare head shape and
pronotum shape among populations. CV axes allow us
to maximize the difference in shape among populations relative to within-population variance. A full crossvalidation discriminate functions analysis (DFA) was
used to determine how well populations are distinguished from each other based on head and pronotum
shape. Males and females were analysed separately.
RESULTS
PHYLOGENETIC ANALYSES
Results from the phylogenetic analyses showed several
distinct evolutionary lineages within S. petersi. Within
S. petersi, two well-supported major clades were identified (clades A and B, Fig. 3); however, shallower clades
had low Bayesian posterior probabilities and/or
maximum-likelihood bootstrap support. The two major
clades of S. petersi corresponded to geographic relationships between collection localities and spatially
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
MORPHOMETRICS OF SCAPHINOTUS PETERSI
5
Figure 3. Maximum-likelihood tree of Scaphinotus petersi populations from combined 28S rDNA, COI, and ND1 + mtRNA
data. The out-group, Sphaeroderus lecontei, is removed to show greater detail. Specimen numbers are removed, but the
subspecies and mountain range from which they were collected is indicated. Specimens from all subspecies in Table 1
are represented in the molecular phylogeny. Support for major branches is indicated by Bayesian posterior probability/
maximum likelihood bootstrap values. *Bayesian posterior probability greater than 95%. Scale bar units are substitutions per site.
structured genetic variation at deep and shallow scales.
Clade A (Fig. 3) of Scaphinotus petersi grahami Van
Dyke, 1938 from the Pinaleño Mountains, Scaphinotus
petersi corvus Fall, 1910 from the Chiricahua Mountains, and Scaphinotus petersi kathleenae Ball, 1966
from the Santa Rita Mountains, was clearly
phylogenetically distinct from clade B of Scaphinotus
petersi catalinae Van Dyke, 1924 from the Santa
Catalina Mountains, Scaphinotus petersi biedermani
Roeschke, 1907 from the Huachuca Mountains,
S. p. biedermani from the Rincon Mountains, and
Scaphinotus petersi petersi Roeschke, 1907 from the
Pinal Mountains (Fig. 3). Scaphinotus petersi kathleenae
from the Santa Rita Mountains was paraphyletic with
respect to other populations in clade A. In clade B, the
S. p. biedermani Rincon population appeared to be a
distinct lineage from the S. p. biedermani Huachuca
population, and within the Huachuca population there
were some lineages more closely related to the Santa
Catalina population. The Santa Catalina population
(S. p. catalinae) was paraphyletic with respect to some
S. p. biedermani from the Huachuca Mountains. Both
maximum-likelihood and Bayesian analyses of combined nuclear and mitochondrial DNA found similar
topologies. The best maximum-likelihood tree (Fig. 3)
had a log-likelihood score of −7347.2245, and the Bayesian analyses converged on a set of trees with a mean
log-likelihood score of −6118.129.
MORPHOMETRIC ANALYSES
The results of the quantitativie morphometrics analyses showed there was a large amount of morphological variation and divergence in traits among S. petersi
populations. Results of ANOVA tests indicated that head
length and width, leg length, and total body length
varied significantly among montane populations for
females (P ≤ 0.040; Fig. 4; Table 2) and males (P ≤ 0.010;
Fig. 4; Table 2), but that there was substantial overlap
among some populations (Table 2). When montane populations were combined into subspecies categories, head
length and width and total body length varied significantly among subspecies of S. petersi for both sexes
(P ≤ 0.009), but there was not significant variation
among subspecies in leg length for males (P = 0.074),
and nor for females (P = 0.112).
Using multivariate analyses of distance measurements we found significant differences among S. petersi
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
6
K. A. OBER AND C. T. CONNOLLY
Figure 4. ANOVA of male length and width trait measurements by mountain range and subspecies: A, male head width;
B, male body length; C, male leg length; D, male head length; E, female head width; F, female body length; G, female
leg length; H, female head length. Black string, median; open box, first interquartile; bar, second interquartile.
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
Catalina
Chiricahua
Huachuca
Rincon
Pinal
Pinaleño
White
Santa Rita
Mountain range
Catalina
Chiricahua
Huachuca
Rincon
Pinal
Sierra Ancha
Pinaleño
White
Santa Rita
catalinae
corvus
biedermani
biedermani
petersi
grahami
grahami
kathleenae
Subspecies
catalinae
corvus
biedermani
biedermani
petersi
petersi
grahami
grahami
kathleenae
52–55
20–21
6–7
2–3
4–6
2
27
4
3–5
No. males
27–30
24
5–7
0–3
6–7
31–33
5
1–3
No. females
Head width (SD)
2.073A,B (0.092)
2.086A,B (0.064)
2.163A (0.070)
2.066A,B (0.143)
2.132A,B (0.130)
1.999A,B (0.016)
2.041B (0.071)
2.055A,B (0.127)
2.144A,B (0.109)
1.906B (0.097)
1.918B (0.083)
2.056A (0.101)
1.944A,B (0.071)
1.925A,B (0.114)
1.855A,B (0.017)
1.912B (0.073)
1.828B (0.069)
2.007A,B (0.077)
2.120A,B (0.092)
2.098A,B (0.083)
2.220A (0.103)
2.084A,B,C (0.109)
2.114A,B,C (0.216)
2.088B (0.085)
2.039A,B,C (0.052)
2.123A,B,C (0.084)
1.994B (0.119)
1.967B (0.085)
2.146A (0.099)
1.921B,C (0.151)
2.051A,B (0.109)
1.981B (0.090)
1.938B (0.094)
1.928A,B,C (0.049)
Head length (SD)
Head width (SD)
Head length (SD)
15.305B (1.061)
15.294A,B (0.612)
16.706A (0.425)
15.006A,B (0.064)
14.867A,B (0.542)
15.236A,B (NA)
15.715A,B (0.836)
14.868B (0.542)
15.879A,B (2.187)
Leg length (SD)
15.966A,B (1.275)
15.319B,C,D (0.952)
16.589A,B,C (1.079)
NA
15.991A,B,C,D (1.390)
16.190A (0.816)
14.147D (0.344)
17.535A,B,C,D (NA)
Leg length (SD)
15.029A,B (0.711)
14.840A,B (0.541)
15.589A (0.468)
14.783A,B (0.373)
14.922A,B (0.364)
14.627B (0.227)
14.975A,B (0.648)
15.194A,B (0.706)
15.708A,B (0.359)
Total body length (SD)
16.197A (1.039)
15.662A,B (0.718)
16.541A (1.060)
NA
16.050A,B (0.908)
16.133A (0.751)
15.861A,B (0.535)
15.800A,B (NA)
Total body length (SD)
Males and females were analysed separately. Averages are reported in mm with the standard deviation (SD) in parentheses. The superscript numbers indicate
the results of the Tukey–Kramer highly significant difference (HSD) test, with populations sharing the same letter not significantly different.
Mountain range
Subspecies
Table 2. Results of linear morphometric measurements for Scaphinotus petersi beetles
MORPHOMETRICS OF SCAPHINOTUS PETERSI
7
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K. A. OBER AND C. T. CONNOLLY
populations in head and pronotum shape. The MANOVA
of distances between landmarks along the head outline
detected significant differences in head shape among
populations and subspecies (Wilk’s Lambda P ≤ 0.003)
for females and males. The ‘within canonical structure’ values in the discriminant analyses above were
greater than |0.4| for the anterior width of the head
and the anterior–posterior position of the eyes on the
head for both males and females, indicating that these
features of head shape were significantly different among
groups. There were significant differences from the
MANOVA among the populations and subspecies of
S. petersi in distances between landmarks along the
pronotum outline (Wilk’s lambda P ≤ 0.009) for males
and females. The anterolateral angles and the
posterolateral angles of the pronota showed significant differences among populations and subspecies with
‘within canonical structure’ values above |0.4|.
The first and second principal components in females
explained 43.76 and 18.87%, respectively, of shape variation in heads and 41.57 and 18.97%, respectively, of
shape variation in pronota. For males, first and second
principal components explained 45.13 and 19.59%, respectively, of shape variation in heads, and 34.57 and
20.37%, respectively, of shape variation in pronota. PC1
for head shape was related to the width of the posterior region of the head and PC2 was related to the
position of the eyes. PC1 and PC2 for pronotum shape
were related to the size and sharpness of the
anterolateral and posterolateral angles, and narrowness of the pronotum. A scatter plot of the first two
principal components revealed no discrete morphological clusters for heads or pronota for either sex (data
not shown); however, there was a great deal of variation in S. petersi across populations and subspecies
in each sex for head shape and pronotum shape.
Discrimination among groups can be interpreted by
examining the ordination of specimens in the
morphospace defined by the CV axes of the partial warp
shape variables. The first two CV axes for heads accounted for 70.11% of the total shape variation in
females, and 83.54% in males. The CVA indicated significant differences among mountain ranges, especially in the pronotum width and the sharpness of the
posterolateral angles (Fig. 5). Based on the permutation tests of 100 000 rounds for Procrustes distances,
the pronotum shape was significantly different
(P ≤ 0.039) for all montane populations in females, except
for the Catalina and Huachuca populations (P = 0.096)
and the Rincon versus Catalina, Pinal, Santa Rita, and
Huachuca (P = 0.053–0.11). Male pronotum shape was
significantly different (P ≤ 0.04) among all montane
populations except for the Rincon and Huachuca populations (both populations of S. p. biedermani, P = 0.24).
The CVA showed significant differences in head shape
in some, but not all, montane populations for both sexes
(Fig. S1; Table 3). There was variation in head shape
among populations and subspecies, but no clear pattern
was discernable.
The DFA of pronotum shape indicated that male and
female beetle specimens can be linked to their mountain range fairly accurately: more than 90.4% of male
beetles and 89.0% of female beetles were correctly assigned based on pronotum shape. If the population
sample size was greater than ten specimens, more than
95% of specimens were correctly assigned. DFA found
significant differences between means in Procrustes distances (P ≤ 0.039) for most populations. A drop in the
accuracy of discrimination was observed in Pinal, Santa
Rita, Rincon, and White Mountain populations as a
result of the small sample sizes (3–7) in these groups.
Head shape did not easily discriminate among specimens from different mountain ranges: 77.0% of male
beetles and 76.8% of female beetles were correctly assigned to their montane population based on head shape
(Table 3).
DISCUSSION
In this study, we recovered a general pattern of genetic
and morphological divergence among several S. petersi
montane populations; however, some populations showed
closer molecular evolutionary relationships and overlaps in morphological traits. Morphological differences between montane populations are weak overall,
but our results show that there are at least two clear
morphologically and genetically distinct groups within
S. petersi. Phylogenetic analyses of molecular sequence data for S. petersi show relationships congruent with the phylogenies of Ball (1966) and Mitchell
& Ober (2013). Populations are grouped into two fairly
well-supported clades: clade A, composed of populations from mostly east of the San Pedro River; and
clade B, composed of populations from west of the San
Pedro River. Relaxed molecular clock divergence dating
by Mitchell & Ober (2013) indicates beetles in these
clades have been separated for about 60 000 years.
Scaphinotus petersi kathleenae from the Santa Ritas
is paraphyletic with respect to other populations east
of the San Pedro River in clade A. Mitchell & Ober
(2013) uncovered weak evidence that the Santa Rita
population may have been the ancestral population for
the other populations in clade A and for a holocene dispersal from the Catalina population to the Huachucas.
In this phylogenetic tree, S. p. biedermani appears
to be polyphyletic, suggesting that samples from the
Huachucas are more closely related to beetles from the
Catalinas than from the Rincons; however, the relationships within clade B are not well supported. The
two S. biedermani populations differed significantly in
head length in females, and to some extent in the head
shape in males. Pronotum shapes for each of these two
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
MORPHOMETRICS OF SCAPHINOTUS PETERSI
9
Figure 5. Scatter plots of canonical variate analyses (CVA) for pronotum shape: CV1 versus CV2 of (A) female and (B)
male pronota. Legend indicates the mountain ranges from where the specimen was collected. For both plots, shape deformation of pronotum is shown for the extreme points of each axis. A dotted line separates populations in clade A of the
phylogenetic tree from those in clade B.
populations of S. p. biedermani appear to occupy different morphospaces based on CVA (Fig. 5), but the
sample sizes are too small to say unequivocally. Although the evidence is not strong, the phylogenetic relationships, morphometrics, and geography suggest
beetles from the Huachuca population and the Rincon
population should not be combined into the same subspecies of S. p. biedermani. Patterns of genetic variation and morphological differences observed in this
study suggest the current taxonomic classification of
S. petersi subspecies may not accurately reflect evolutionary relationships.
In addition to Ball (1966), several other studies of
Arizona Sky Island organisms have found significant
morphological divergence among montane populations, including land snails in the genus Sonorella
(Miller, 1967; Bequaert & Miller, 1973), jumping spiders
Habronattus pugillis Griswold, 1987 (Maddison &
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
10
K. A. OBER AND C. T. CONNOLLY
Table 3. Results of canonical variate analyses (CVA) and discriminate functions analysis (DFA) comparing geometric
morphometric differences in Scaphinotus petersi head shape
Females
Chiricahua
Huachuca
Pinal
Pinaleño
Rincon
Santa Rita
White
Catalina
Chiricahua
Huachuca
Pinal
Pinaleño
< 0.0001
< 0.0001
0.2140
0.0140
0.4272
0.2076
< 0.0001
< 0.0001
0.6320
0.0211
0.2342
0.2893
0.0074
0.0993
0.0569
0.0093
0.2606
0.1203
0.0022
< 0.0001
0.0594
0.0012
0.1063
0.2686
0.0144
0.0127
0.8348
0.3340
0.0550
0.1198
0.4428
0.6603
0.1691
0.8357
0.0304
0.4094
0.4061
0.4508
0.8181
0.6500
0.2821
0.6860
0.0584
0.4387
Catalina
Chiricahua
Huachuca
Pinal
Pinaleño
0.3448
< 0.0001
0.2045
0.0026
0.8742
0.1597
0.0411
< 0.0001
0.3370
0.0126
0.100
0.0006
0.2487
0.3631
0.2495
0.0195
0.0092
0.0248
0.0988
0.1624
< 0.0001
< 0.0001
0.0601
0.0012
0.0340
0.0741
0.1476
0.0061
0.0270
0.3007
0.0826
0.2301
0.974
0.8875
0.0827
0.2913
0.0055
0.3922
0.2077
0.6653
0.1085
0.3958
0.0188
0.0316
0.4521
0.9074
0.0102
0.5599
0.6284
0.9311
0.1950
0.6838
0.0241
0.0406
< 0.0001
0.0001
0.0901
0.2975
0.0447
0.1566
0.2954
0.2311
0.0098
0.0009
0.0003
0.0011
Rincon
Santa Rita
< 0.0001
0.7939
< 0.0001
0.7275
0.2866
0.7751
Rincon
Santa Rita
Sierra Ancha
0.0192
0.7595
0.0153
0.2832
0.1099
0.6638
Males
Chiricahua
Huachuca
Pinal
Pinaleño
Rincon
Santa Rita
Sierra Ancha
White
0.0583
0.2263
0.6004
0.8305
0.1399
0.3970
Upper values are P values from CVA permutation tests (100 000 permutation rounds) for Procrustes distances between
populations. Lower values are P values from DFA difference between means of Procrustes distances. Numbers in bold
indicate that head shape significantly differs between populations. Females and males were analysed separately.
McMahon, 2000), scorpions in the genus Vaejovis
(Hughes, 2011), indian paintbrush plants Castilleja
austromontana (Slentz, Boyd & McDade, 1999), and
giant-trumpets Macromeria viridiflora (Boyd, 2002). Concordant biogeographic patterns can be seen in populations of organisms distributed on the Sky Islands.
Ball (1966), Masta (2000), Boyd (2002), McCormack,
Bowen & Smith (2008), and Hughes (2011) also reported a north–south mountain range relationship
among populations with an east–west gap. This pattern
is also reflected in the phylogeography of populations
of S. petersi.
Statistical analyses of morphological differences in
S. petersi showed significant differences in body size
and head and pronotum shape among several populations and subspecies, as noted by Ball (1966); however,
the overall pattern seen with the data presented here
is conflicting and difficult to discern. For instance, the
linear measurements of leg, body, head length, head
width, and head shape did not consistently
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015
MORPHOMETRICS OF SCAPHINOTUS PETERSI
correspond to named subspecies, geographic distance
between montane populations, or clades identified using
molecular data. Additionally, small sample sizes in some
populations (Pinal, Sierra Ancha, Santa Rita, Rincon,
and White Mountains) made it difficult to detect significant differences. In S. p. petersi, males from the
Sierra Anchas in the north were the smallest for most
traits except leg length, followed by other northern populations such as the White Mountains etc. It should be
noted that S. petersi is sympatric with Scaphinotus
vandykei Roeschke, 1907 in the Sierra Anchas, but is
easily distinguished by features of the prontotum. Male
beetles from the southernmost population of
S. p. biedermani in the Huachucas were the largest for
most traits, followed by other southern populations,
such as the Santa Rita population, etc. Although trait
length measurements did not distinguish evolutionary clades or discrete populations, there seems to be
a trend in the geographic pattern of larger beetles in
the south and smaller beetles in the north for males.
These results differ slightly from thos of Ball (1966),
where he reported the largest total body length in the
Pinals (N = 23–32) and smallest total body length in
the Chiricahuas (N = 15–21); however, Ball also found
a large overlap in trait lengths and widths among populations. Females did not show a clear north–south size
trend in any of the morphological traits. Although there
are significant differences among some populations and
subspecies in head shape, they do not clearly follow
the clade A and clade B pattern, or a north–south geographical pattern. The six landmarks chosen to describe head shape may not have captured the subtlety
of head shape differences among populations; however,
the size of the head (length and width) and the anterior–
posterior position of the eyes seemed to vary significantly among some populations. For instance, the
Huachuca population was significantly larger than most
other populations.
This study showed the pronotum shape generally reflects phylogenetic relationships, and may be the most
important morphological trait for recognizing distinct populations of S. petersi in the Arizona Sky Islands.
There was a clear pronotum shape distinction between
beetles in clade A and clade B (Fig. 5). In a CVA using
pronotum shape, specimens from clades A and B largely
occupied different regions of morphospace, with little
overlap between them. Beetles in clade A had a narrower pronotum with sharper hind angles than beetles
in clade B. There was also a geographical pattern in
pronotum shape, with northern populations having
slightly narrower pronota within clades A and B. The
distinct lineages of clade A and clade B have evolved
genetic and morphological differences through drift or
selection (natural or sexual). The most rapidly evolving region of the pronotum appeared to be the anteroand posterolateral angles. Female pronotum shape is
11
presumably important for mating, as male S. petersi
hold onto the female near the posterolateral region of
her pronotum with expanded fore tarsi with specialized setae (Stork & Evans, 1976). In doing so, he contacts the lateral and anterolateral edges of her prontoum
with his antennae (K. A. Ober, pers. observ.), suggesting that pronotum shape may be under sexual selection, although there is no direct evidence to support
this hypothesis. We propose that if genetic and geographical isolation continue over time, these populations may eventually evolve into separate species;
however, most Arizona Sky Island S. petersi populations are facing possible extirpation in light of the predicted global warming arising from climate change
(Mitchell & Ober, 2013).
ACKNOWLEDGEMENTS
The authors thank Kipling Will and Robert Davidson
for allowing us to study specimens in their care. We
thank David Kavanaugh for images of some of the specimens in this study. Abby Drake and Justin McAllister
provided advice and help with some morphometric
analyses. Ryan Judy produced several of the 28S rDNA
sequences. We thank the College of the Holy Cross for
funding this project. We are grateful to two anonymous reviewers who provided helpful comments for improving this study and article.
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article at the publisher’s web-site:
Figure S1. Scatterplots of head shape CVA: CV1 versus CV2 of (A) female and (B) male heads. Legend indicates mountain range where specimen was collected.
Table S1. Specimens, collection localities, and GenBank numbers for molecular phylogenetic analyses included in this study. NA indicates DNA sequence not available and * indicates sequences from Mitchell & Ober
(2013).
© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015