Bone Inner Structure Suggests Increasing Aquatic Adaptations in Desmostylia (Mammalia,Afrotheria)

Background

The paleoecology of desmostylians has been discussed controversially with a general consensus that desmostylians were aquatic or semi-aquatic to some extent. Bone microanatomy can be used as a powerful tool to infer habitat preference of extinct animals. However, bone microanatomical studies of desmostylians are extremely scarce.

Methodology/Principal Findings

We analyzed the histology and microanatomy of several desmostylians using thin-sections and CT scans of ribs, humeri, femora and vertebrae. Comparisons with extant mammals allowed us to better understand the mode of life and evolutionary history of these taxa. Desmostylian ribs and long bones generally lack a medullary cavity. This trait has been interpreted as an aquatic adaptation among amniotes. Behemotops and Paleoparadoxia show osteosclerosis (i.e. increase in bone compactness), and Ashoroa pachyosteosclerosis (i.e. combined increase in bone volume and compactness). Conversely, Desmostylus differs from these desmostylians in displaying an osteoporotic-like pattern.

Conclusions/Significance

In living taxa, bone mass increase provides hydrostatic buoyancy and body trim control suitable for poorly efficient swimmers, while wholly spongy bones are associated with hydrodynamic buoyancy control in active swimmers. Our study suggests that all desmostylians had achieved an essentially, if not exclusively, aquatic lifestyle. Behemotops, Paleoparadoxia and Ashoroa are interpreted as shallow water swimmers, either hovering slowly at a preferred depth, or walking on the bottom, and Desmostylus as a more active swimmer with a peculiar habitat and feeding strategy within Desmostylia. Therefore, desmostylians are, with cetaceans, the second mammal group showing a shift from bone mass increase to a spongy inner organization of bones in their evolutionary history.     

Swimming Mode Inferred from Skeletal Proportions in the Fossil Pinnipeds Enaliarctos and Allodesmus (Mammalia, Carnivora)

Swimming modes are crucial for understanding evolutionary transitions from land to sea, because locomotion affects many aspects of an animal’s life. The modern pinniped families Otariidae (fur seals and sea lions), Phocidae (true seals), and Odobenidae (walruses) are thought to share a common origin, but each differs in its primary mode of aquatic locomotion. Previous studies of locomotor evolution in pinnipeds suggested: (1) forelimb swimming was ancestral; (2) hind limb swimming evolved once at the base of the clade including Phocidae, Odobenidae, and the extinct Desmatophocidae; and (3) reversal to forelimb swimming occurred in the odobenid subfamily Dusignathinae. The oldest and most basal pinnipedimorph Enaliarctos mealsi has been portrayed as a forelimb swimmer, and the desmatophocid Allodesmus kelloggi has been portrayed as a hind limb swimmer. These interpretations have been questioned by others and are tested here. Principal components analysis of trunk and limb measurements from 58 modern semiaquatic mammals demonstrates that Enaliarctos is most similar in skeletal proportions to hind limb-dominated swimmers, whereas Allodesmus is most similar to forelimb-dominated swimmers. Principal components and discriminant function analyses of trunk and limb measurements from 24 modern pinniped species demonstrate that Enaliarctos is most similar to hind limb-swimming phocids, while Allodesmus is most similar to forelimb-swimming otariids. These interpretations complicate previous portrayals of swimming evolution in pinnipeds and can paint a very different picture of how this behavior evolved when viewed in the context of alternative phylogenetic hypotheses.     

Site-specific fatty acid composition in adipose tissues of several northern aquatic and terrestrial mammals

Site-specific differences in fatty acid compositions (by gas-liquid chromatography) were compared in aquatic, semiaquatic and terrestrial mammals: the ringed seals (Phoca hispida hispida and P. h. botnica), otter (Lutra lutra), raccoon dog (Nyctereutes procyonoides), brown bear (Ursus arctos) and grey wolf (Canis lupus). In addition, we briefly discuss our earlier results for the Canadian beaver (Castor canadensis) and muskrat (Ondatra zibethicus). In both aquatic and terrestrial species, large amounts of Δ9-monounsaturated fatty acids (MUFAs) and small amounts of saturated fatty acids and exogenous long-chain MUFAs were found in the cold tissues of the extremities. In seals, the poikilothermic outer blubber had these characteristics and differed from the inner blubber. On the other hand, the subcutaneous and inner fat depots of the coated semiaquatic and terrestrial mammals were uniform. In the bare extremities, however, these mammals also had an excess of A9-MUFAs. The degree of Δ9-desaturation in the outer blubber of the seals was significantly correlated with age. The excess of Δ9-MUFAs in the bare extremities of land mammals increased the overall double bond content of these tissues compared with the inner depots. In contrast, due to the large amounts of dietary polyunsaturated fatty acids, this was not found in the aquatic and semiaquatic species. The observed site-specific differences are discussed as possible inherited evolutionary adaptations to low temperature of the tissues.     

Olecranon orientation as an indicator of elbow joint angle in the stance phase,and estimation of forelimb posture in extinct quadruped animals

Reconstruction of limb posture is a challenging task in assessing functional morphology and biomechanics of extinct tetrapods, mainly because of the wide range of motions possible at each limb joint and because of our poor knowledge of the relationship between posture and musculoskeletal structure, even in the extant taxa. This is especially true for extinct mammals such as the desmostylian taxa Desmostylus and Paleoparadoxia. This study presents a procedure that how the elbow joint angles of extinct quadruped mammals can be inferred from osteological characteristics. A survey of 67 dried skeletons and 113 step cycles of 32 extant genera, representing 25 families and 13 orders, showed that the olecranon of the ulna and the shaft of the humerus were oriented approximately perpendicular to each other during the stance phase. At this angle, the major extensor muscles maximize their torque at the elbow joint. Based on this survey, I suggest that olecranon orientation can be used for inferring the elbow joint angles of quadruped mammals with prominent olecranons, regardless of taxon, body size, and locomotor guild. By estimating the elbow joint angle, it is inferred that Desmostylus would have had more upright forelimbs than Paleoparadoxia, because their elbow joint angles during the stance phase were approximately 165° and 130°, respectively. Difference in elbow joint angles between these two genera suggests possible differences in stance and gait of these two mammals. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Relationship between osteology and aquatic locomotion in birds: determining modes of locomotion in extinct Ornithurae

The evolutionary history of aquatic invasion in birds would be incomplete without incorporation of extinct species. We show that aquatic affinities in fossil birds can be inferred by multivariate analysis of skeletal features and locomotion of 245 species of extant birds. Regularized discriminant analyses revealed that measurements of appendicular skeletons successfully separated diving birds from surface swimmers and flyers, while also discriminating among different underwater modes of swimming. The high accuracy of this method allows detection of skeletal characteristics that are indicative of aquatic locomotion and inference of such locomotion in bird species with insufficient behavioural information. Statistical predictions based on the analyses confirm qualitative assessments for both foot‐propelled (Hesperornithiformes) and wing‐propelled (Copepteryx) underwater locomotion in fossil birds. This is the first quantitative inference of underwater modes of swimming in fossil birds, enabling future studies of locomotion in extinct birds and evolutionary transitions among locomotor modes in avian lineage.     

Distal Humeral Morphology Indicates Locomotory Divergence in Extinct Giant Kangaroos

Previous studies of the morphology of the humerus in kangaroos showed that the shape of the proximal humerus could distinguish between arboreal and terrestrial taxa among living mammals, and that the extinct “giant” kangaroos (members of the extinct subfamily Sthenurinae and the extinct macropodine genus Protemnodon) had divergent humeral anatomies from extant kangaroos. Here, we use 2D geometric morphometrics to capture the shape of the distal humerus in a range of extant and extinct marsupials and obtain similar results: sthenurines have humeral morphologies more similar to arboreal mammals, while large Protemnodon species (P. brehus and P. anak) have humeral morphologies more similar to terrestrial quadrupedal mammals. Our results provide further evidence for prior hypotheses: that sthenurines did not employ a locomotor mode that involved loading the forelimbs (likely employing bipedal striding as an alternative to quadrupedal or pentapedal locomotion at slow gaits), and that large Protemnodon species were more reliant on quadrupedal locomotion than their extant relatives. This greater diversity of locomotor modes among large Pleistocene kangaroos echoes studies that show a greater diversity in other aspects of ecology, such as diet and habitat occupancy.

     

Skeletal indicators of locomotor adaptations in living and extinct rodents

Living rodents show great diversity in their locomotor habits, including semiaquatic, arboreal, fossorial, ricochetal, and gliding species from multiple families. To assess the association between limb morphology and locomotor habits, the appendicular skeletons of 65 rodent genera from 16 families were measured. Ecomorphological analyses of various locomotor types revealed consistent differences in postcranial skeletal morphology that relate to functionally important traits. Behaviorally similar taxa showed convergent morphological characters, despite distinct evolutionary histories. Semiaquatic rodents displayed relatively robust bones, enlarged muscular attachments, short femora, and elongate hind feet. Arboreal rodents had relatively elongate humeri and digits, short olecranon processes of the ulnae, and equally proportioned fore and hind limbs. Fossorial rodents showed relatively robust bones, enlarged muscular attachments, short antebrachii and digits, elongate manual claws, and reduced hind limb elements. Ricochetal rodents displayed relatively proximal insertion of muscles, disproportionate limbs, elongate tibiae, and elongate hind feet. Gliding rodents had relatively elongate and gracile bones, short olecranon processes of the ulnae, and equally proportioned fore and hind limbs. The morphological differences observed here can readily be used to discriminate extant rodents with different locomotor strategies. This suggests that the method could be applied to extinct rodents, regardless of ancestry, to accurately infer their locomotor ecologies. When applied to an extinct group of rodents, we found two distinct ecomorphs represented in the beaver family (Castoridae), semiaquatic and semifossorial. There was also a progressive trend toward increased body size and increased aquatic specialization in the giant beaver lineage (Castoroidinae). J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Postcranial morphology and the locomotor habits of living and extinct carnivorans

Members of the order Carnivora display a broad range of locomotor habits, including cursorial, scansorial, arboreal, semiaquatic, aquatic, and semifossorial species from multiple families. Ecomorphological analyses from osteological measurements have been used successfully in prior studies of carnivorans and rodents to accurately infer the locomotor habits of extinct species. This study uses 20 postcranial measurements that have been shown to be effective indicators of locomotor habits in rodents and incorporates an extensive sample of over 300 individuals from more than 100 living carnivoran species. We performed statistical analyses, including analysis of variance (ANOVA) and stepwise discriminant function analysis, using a set of 16 functional indices (ratios). Our ANOVA results reveal consistent differences in postcranial skeletal morphology among locomotor groups. Cursorial species display distal elongation of the limbs, gracile limb elements, and relatively narrow humeral and femoral epicondyles. Aquatic and semiaquatic species display relatively robust, shortened femora and elongate metatarsals. Semifossorial species display relatively short, robust limbs with enlarged muscular attachment sites and elongate claws. Both semiaquatic and semifossorial species have relatively elongate olecranon process of the ulna and enlarged humeral and femoral epicondyles. Terrestrial, scansorial, and arboreal species are characterized by having primarily intermediate features, but arboreal species do show relatively elongate manual digits. Morphological indices effectively discriminate locomotor groups, with cursorial and arboreal species more accurately classified than terrestrial, scansorial, or semiaquatic species. Both within and between families, species with similar locomotor habits converge toward similar postcranial morphology despite their independent evolutionary histories. The discriminant analysis worked particularly well to correctly classify members of the Canidae, but not as well for members of the Mustelidae or Ursidae. Results are used to infer the locomotor habits of extinct carnivorans, including members of several extinct families, and also 12 species from the Pleistocene of Rancho La Brea. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Swimming performance in semiaquatic and terrestrial Oryzomyine rodents

Semiaquatic and terrestrial mammals frequently have to cross or move along water bodies, both trying to remain on the water surface using one or two pairs of limbs, combining different gaits and stride lengths and frequencies. This is the case of the semiaquatic water rats Nectomys and the cursorial Cerradomys, sister genera of the Oryzomyini tribe, capable of swimming using similar gaits. They provide an opportunity to investigate performance specializations involving the semiaquatic habitat, our objective in this study. Rodents were filmed at 30 frames s⁻¹ in lateral view, swimming in a glass aquarium. Video sequences were analyzed dividing the swimming cycle into power and recovery phases. Differences in swimming performance were detected between species of Nectomys and Cerradomys, but not between species of the same genus. Absolute mean speed did not differ between the semiaquatic and terrestrial groups, but the semiaquatic Nectomys had longer stride lengths with lower stride frequency, whereas the terrestrial Cerradomys had higher stride frequency and relative swimming speed. The widest behavior repertoire of Nectomys allowed more efficient, but not necessarily faster swimming than the terrestrial Cerradomys. Efficient aquatic locomotion in Nectomys is ultimately a result of improved buoyancy by hydrophobic fur and subtle morphological specializations, which allow this genus to perform more efficiently in water than the terrestrial Cerradomys without compromising locomotion in the terrestrial environment.     

Evolving between land and water: key questions on the emergence and history of the Hippopotamidae (Hippopotamoidea,Cetancodonta, Cetartiodactyla)

The fossil record of the Hippopotamidae can shed light on three major issues in mammalian evolution. First, as the Hippopotamidae are the extant sister group of Cetacea, gaining a better understanding of the origin of the Hippopotamidae and of their Paleogene ancestors will be instrumental in clarifying phylogenetic relationships within Cetartiodactyla. Unfortunately, the data relevant to hippopotamid origins have generally been ignored in phylogenetic analyses of cetartiodactyls. In order to obtain better resolution, future analyses should consider hypotheses of hippopotamid Paleogene relationships. Notably, an emergence of the Hippopotamidae from within anthracotheriids has received growing support, leading to reconciliation between genetic and morphological evidence for the clade Cetancodonta (Hippopotamidae + Cetacea). Secondly, full account needs to be taken of the Hippopotamidae when studying the impact of environmental change on faunal evolution. This group of semi‐aquatic large herbivores has a clear and distinct ecological role and a diverse and abundant fossil record, particularly in the African Neogene. We examine three major phases of hippopotamid evolution, namely the sudden appearance of hippopotamines in the late Miocene (the “Hippopotamine Event”), the subsequent rampant endemism in African basins, and the Pleistocene expansion of Hippopotamus. Each may have been influenced by multiple factors, including: late Miocene grass expansion, African hydrographical network disruption, and a unique set of adaptations that allowed Hippopotamus to respond efficiently to early Pleistocene environmental change. Thirdly, the fossil record of the Hippopotamidae documents the independent emergence of adaptive character complexes in relation to semiaquatic habits and in response to insular isolation. The semiaquatic specializations of fossil hippopotamids are particularly useful in interpreting the functional morphology and ecology of other, extinct groups of large semiaquatic herbivores. Hippopotamids can also serve as models to elucidate the evolutionary dynamics of island mammals.     

Salient features in the locomotion of proboscideans revealed via the differential scaling of limb long bones

The standard differential scaling of proportions in limb long bones (length against circumference) was applied to a phylogenetically wide sample of the Proboscidea, Elephantidae and the Asian (Elephas maximus) and African (Loxodonta africana) elephants. In order to investigate allometric patterns in proboscideans and terrestrial mammals with parasagittal limb kinematics, the computed slopes between long bone lengths and circumferences (slenderness exponents) were compared with published values for mammals, and studied within a framework of the theoretical models of long bone scaling under gravity and muscle forces. Limb bone allometry in E. maximus and the Elephantidae is congruent with adaptation to bending and/or torsion induced by muscular forces during fast locomotion, as in other mammals, whereas the limb bones in L. africana appear to be adapted for coping with the compressive forces of gravity. Hindlimb bones are therefore more compliant than forelimb bones, and the resultant limb compliance gradient in extinct and extant elephants, contrasting in sign to that of other mammals, is shown to be a new important locomotory constraint preventing elephants from achieving a full‐body aerial phase during fast locomotion. Moreover, the limb bone pattern of African elephants, indicating a noncritical bone stress not increasing with increments in body weight, explains why their mean and maximal body masses are usually above those for Asian elephants. Differences in ecology may be responsible for the subtle differences observed in vivo between African and Asian elephants, but they appear to be more pronounced when revealed via mechanical patterns dictated by limb bone allometry. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 16–29.     

Trabecular Architecture of the Manual Elements Reflects Locomotor Patterns in Primates

The morphology of trabecular bone has proven sensitive to loading patterns in the long bones and metacarpal heads of primates. It is expected that we should also see differences in the manual digits of primates that practice different methods of locomotion. Primate proximal and middle phalanges are load-bearing elements that are held in different postures and experience different mechanical strains during suspension, quadrupedalism, and knuckle walking. Micro CT scans of the middle phalanx, proximal phalanx and the metacarpal head of the third ray were used to examine the pattern of trabecular orientation in Pan, Gorilla, Pongo, Hylobates and Macaca. Several zones, i.e., the proximal ends of both phalanges and the metacarpal heads, were capable of distinguishing between knuckle-walking, quadrupedal, and suspensory primates. Orientation and shape seem to be the primary distinguishing factors but differences in bone volume, isotropy index, and degree of anisotropy were seen across included taxa. Suspensory primates show primarily proximodistal alignment in all zones, and quadrupeds more palmar-dorsal orientation in several zones. Knuckle walkers are characterized by having proximodistal alignment in the proximal ends of the phalanges and a palmar-dorsal alignment in the distal ends and metacarpal heads. These structural differences may be used to infer locmotor propensities of extinct primate taxa.     

Intrinsic hand proportions of euarchontans and other mammals: implications for the locomotor behavior of plesiadapiforms

Arboreal primates have distinctive intrinsic hand proportions compared with many other mammals. Within Euarchonta, platyrrhines and strepsirrhines have longer manual proximal phalanges relative to metacarpal length than colugos and terrestrial tree shrews. This trait is part of a complex of features allowing primates to grasp small-diameter arboreal substrates. In addition to many living and Eocene primates, relative elongation of proximal manual phalanges is also present in most plesiadapiforms. In order to evaluate the functional and evolutionary implications of manual similarities between crown primates and plesiadapiforms, we measured the lengths of the metacarpal, proximal phalanx, and intermediate phalanx of manual ray III for 132 extant mammal species (n=702 individuals). These data were compared with measurements of hands in six plesiadapiform species using ternary diagrams and phalangeal indices. Our analyses reveal that many arboreal mammals (including some tree shrews, rodents, marsupials, and carnivorans) have manual ray III proportions similar to those of various arboreal primates. By contrast, terrestrial tree shrews have hand proportions most similar to those of other terrestrial mammals, and colugos are highly derived in having relatively long intermediate phalanges. Phalangeal indices of arboreal species are significantly greater than those of the terrestrial species in our sample, reflecting the utility of having relatively long digits in an arboreal context. Although mammals known to be capable of prehensile grips demonstrate long digits relative to palm length, this feature is not uniquely associated with manual prehension and should be interpreted with caution in fossil taxa. Among plesiadapiforms, Carpolestes, Nannodectes, Ignacius, and Dryomomys have manual ray III proportions that are unlike those of most terrestrial species and most similar to those of various arboreal species of primates, tree shrews, and rodents. Within Euarchonta, Ignacius and Carpolestes have intrinsic hand proportions most comparable to those of living arboreal primates, while Nannodectes is very similar to the arboreal tree shrew Tupaia minor. These results provide additional evidence that plesiadapiforms were arboreal and support the hypothesis that Euarchonta originated in an arboreal milieu.     

Relationships of Endemic African Mammals and Their Fossil Relatives Based on Morphological and Molecular Evidence

Analyses of anatomical and DNA sequence data run on a parallel supercomputer that include fossil taxa support the inclusion of tenrecs and golden moles in the Afrotheria, an endemic African clade of placental mammals. According to weighting schemes of morphological and molecular data that maximize congruence, extinct members of the afrotherian crown group include embrithopods, Plesiorycteropus, desmostylians, and the condylarths Hyopsodus, Meniscotherium, and possibly Phenacodus. By influencing the optimization of anatomical characters, molecular data have a large influence on the relationships of several extinct taxa. The inclusion of fossils and morphological data increases support for an elephant-sea cow clade within Paenungulata and identifies ancient, northern elements of a clade whose living members in contrast suggest an historically Gondwanan distribution. In addition, maximally congruent topologies support the position of Afrotheria as well-nested, not basal, within Placentalia. This pattern does not accord with the recent hypothesis that the divergence of placental mammals co-occurred with the tectonic separation of Africa and South America.     

Dietary habits of polar bears in Foxe Basin,Canada: possible evidence of a trophic regime shift mediated by a new top predator

Polar bear (Ursus maritimus) subpopulations in several areas with seasonal sea ice regimes have shown declines in body condition, reproductive rates, or abundance as a result of declining sea ice habitat. In the Foxe Basin region of Nunavut, Canada, the size of the polar bear subpopulation has remained largely stable over the past 20 years, despite concurrent declines in sea ice habitat. We used fatty acid analysis to examine polar bear feeding habits in Foxe Basin and thus potentially identify ecological factors contributing to population stability. Adipose tissue samples were collected from 103 polar bears harvested during 2010–2012. Polar bear diet composition varied spatially within the region with ringed seal (Pusa hispida) comprising the primary prey in northern and southern Foxe Basin, whereas polar bears in Hudson Strait consumed equal proportions of ringed seal and harp seal (Pagophilus groenlandicus). Walrus (Odobenus rosmarus) consumption was highest in northern Foxe Basin, a trend driven by the ability of adult male bears to capture large‐bodied prey. Importantly, bowhead whale (Balaena mysticetus) contributed to polar bear diets in all areas and all age and sex classes. Bowhead carcasses resulting from killer whale (Orcinus orca) predation and subsistence harvest potentially provide an important supplementary food source for polar bears during the ice‐free period. Our results suggest that the increasing abundance of killer whales and bowhead whales in the region could be indirectly contributing to improved polar bear foraging success despite declining sea ice habitat. However, this indirect interaction between top predators may be temporary if continued sea ice declines eventually severely limit on‐ice feeding opportunities for polar bears.     

Functional significance of bone ballastin in the evolution of buoyancy control strategies by aquatic tetrapods

The primary function of pachyostosis, pachyosteo‐sclerosis, and osteosclerosis may be to act as ballast, not so much (as previously suggested) to neutralise the buoyancy of existing lungs, but to allow enlargement of the lungs. Enlarged lungs cause an animal to lose buoyancy more rapidly with depth. They also provide a larger oxygen store. These features are useful for slow swimmers and shallow divers, such as feeders on benthic plants and invertebrates. Examples are sirenians, primitive sauropterygians ("not‐hosaurs"), placodonts, and the sea otter Enhydra. These last two show convergent evolution of adaptations to feeding on hard‐shelled invertebrate prey in shallow water. Mesosaurids are problematical. Bone ballast uses body mass and volume less efficiently than other buoyancy control strategies. Theoretical analysis predicts that bone ballast should not occur in semiaquatic forms, fast swimmers or deep divers. It does not usually occur in such organisms. Marine iguanas of the Galápagos, desmostylians, and the aquatic sloth Thalassocnus are all littoral feeders and all lack bone ballast as predicted. Plesiosaurs adopted varied strategies: some used bone ballast, and others used gastroliths. Biomechanical considerations lead to the prediction that a new marine tetrapod clade will typically evolve bone ballast as part of its adaptation to life in water. Slow swimmers and grazers on sessile food, like sirenians and placodonts, develop it more strongly, but active predators like ichthyosaurs and cetaceans secondarily lose this character.     

Observations of a wild polar bear (<Emphasis Type="Italic">Ursus maritimus</Emphasis>) successfully fishing Arctic charr (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>) and Fourhorn sculpin (<Emphasis Type="Italic">Myoxocephalus quadricornis</Emphasis>)

Polar bears, Ursus maritimus, throughout their range, are nutritionally dependent on ringed (Phoca hispida) and bearded seals (Erignathus barbatus), which are predominantly caught on the sea ice. Other marine prey species are caught and consumed, but less frequently. As the annual sea ice retreats, polar bears throughout their range are forced ashore, where they mostly live off their stored adipose tissue. However, while land-bound they have been observed catching birds and terrestrial mammals. Although polar bears evolved from brown bears (U. arctos), direct observations of polar bears diving for and catching fish have not been reported. Here, we document observations of a young male polar bear catching Arctic charr (Salvelinus alpinus) and Fourhorn sculpin (Myoxocephalus quadricornis) by diving in Creswell Bay, Nunavut. We recorded six search bouts, where six fish were caught during dives, which were preceded by a snorkel. The average dive and snorkel length was (mean ± SD) 13 ± 5 and 6 ± 2 s, respectively.