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Computer-integrated polarisation (CIP) in the analysisof fossils: a case of study in a Palaeozoic coral(Sinopora, Syringoporicae, Carboniferous)Ismael Coronadoa, Alberto Pérez-Huertab & Sergio Rodríguezac

a Departamento de Paleontología, Universidad Complutense de Madrid, C/José AntonioNováis 2, Ciudad Universitaria, E-28040 Madrid, Spainb Department of Geological Sciences, University of Alabama, Tuscaloosa, AL 35487, USAc Instituto de Geociencias (IGEO, CSIC-UCM), C/José Antonio Nováis 2, Ciudad Universitaria,E-28040 Madrid, SpainPublished online: 23 Jul 2014.

To cite this article: Ismael Coronado, Alberto Pérez-Huerta & Sergio Rodríguez (2014): Computer-integrated polarisation(CIP) in the analysis of fossils: a case of study in a Palaeozoic coral (Sinopora, Syringoporicae, Carboniferous), HistoricalBiology: An International Journal of Paleobiology, DOI: 10.1080/08912963.2014.938236

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Computer-integrated polarisation (CIP) in the analysis of fossils: a case of study in a Palaeozoiccoral (Sinopora, Syringoporicae, Carboniferous)

Ismael Coronadoa*, Alberto Perez-Huertab and Sergio Rodrıgueza,c

aDepartamento de Paleontologıa, Universidad Complutense de Madrid, C/Jose Antonio Novais 2, Ciudad Universitaria, E-28040Madrid, Spain; bDepartment of Geological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA; cInstituto de Geociencias(IGEO, CSIC-UCM), C/Jose Antonio Novais 2, Ciudad Universitaria, E-28040 Madrid, Spain

(Received 6 May 2014; accepted 21 June 2014)

Computer-integrated polarisation (CIP) method has been applied satisfactorily in the study of fossils skeletons of Sinopora(tabulate coral, Auloporida and Carboniferous). A previous characterisation of sample by scanning electron microscopy,atomic force microscopy and cathodoluminescence (CL) with the purpose of distinguishing the diagenetical alteration wasdone. Subsequently, a crystallographic comparison between CIP and electron-backscattering diffraction has been madegetting a very good correlation between both methods. The CIP method allows obtaining c-axis orientation images, polefigures, and measure and mapping the misorientation of uniaxial biominerals in recent and fossil skeletons. This techniquecan only be used in uniaxial biominerals (calcite, quartz and hydroxylapatite), limiting its use for biaxial or bimineralic andpolimineralic biominerals. CIP method has good spatial resolution (limited by camera); in our example 90 nm. The mainadvantage of this technique, versus other with similar properties, is the fast acquisition of data in low and highmagnifications. This method does not require special treatment of samples and can be very useful for the analysis ofmicrostructures in thin and ultra-thin sections. CIP method detects diagenetic alterations in fossil skeletons by modificationsin the inner arrangement of biominerals, which combined with CL offers valuable geochemical and crystallographicinformation.

Keywords: computer-integrated polarisation; electron-backscattering diffraction; biomineralisation; palaeontology;tabulate corals; Sinopora

Introduction

Matrix-mediated controlled biominerals are hierarchical

structures, with a significant arrangement from nano to

macroscales, to form robust skeletons. This structuring

develops some singular properties at mechanical level,

establishing a research focus for current cutting-edge

biomaterial research (Burdick and Mauck 2011; DiMasi

and Gower 2014). Likewise, these super-structures high-

light the natural evolutionary processes of biomineralisa-

tion that is of interest in palaeontology, particularly the

control exerted by organisms in the formation of skeletons

throughout past environmental and climatic changes.

Calcium carbonate biominerals are an example of such

hierarchical structures with the controlled formation of

crystalline microstructures (Griesshaber et al. 2007;

Beniash 2011). These microstructures have singular

crystallographic properties due to their ordered structure,

representing an evolutionary advantage to organisms for

better adaptation to environments, protection, and improve-

ment of ecological interactions, and overall functional

morphology (Feng et al. 2000; Mann 2001; Schmahl et al.

2004; Currey 2005).

The analysis of the evolution of biomineral properties

can be achieved by the study of fossils (Dauphin 2002;

Stolarski et al. 2007; Cuif et al. 2011; Perez-Huerta et al.

2012). The study of mechanisms responsible for the

development of skeletons in palaeontology is very useful to

understand the common crystallisation pathways in some

groups and their evolution through the time (Stolarski and

Mazur 2005; Cuif et al. 2011). Also, biomineral properties,

such as preferred crystallographic orientations and

geochemical signatures, can help us to distinguish the

systematic affinity of uncertain taxa and to observe the

preservation state of fossils. This last fact is very helpful in

geochemical studies with focus on palaeoclimatic and

palaeoenvironmental interpretations, in which good fossil

preservation may reflect primary chemical signals of

skeletons and shells (Grossman et al. 1996; Mii et al. 2001;

Rosales et al. 2004). Techniques routinely used in the study

of biomineralisation can also be used to inform about the

preservation state of fossils (Perez-Huerta et al. 2007;

Stolarski et al. 2007; Fryda et al. 2009; Grellet-Tinner et al.

2011; Lee et al. 2012; Moreno-Azanza et al. 2013; Torney

et al., 2013).

Classically, the studies of microstructure in palaeozoic

corals have been focused to identify and classify

evolutionary patterns of biocalcification and their

applications as taxonomy criteria. However, this fact has

q 2014 Taylor & Francis

*Corresponding author. Email: ismael.coronado@geo.ucm.es

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been charged of controversy during more than 70 years,

because some authors have considered some or all

microstructures present in palaeozoic corals as a product

of diagenetical alteration (more details in Rodrıguez 1989;

Coronado et al. 2013). By the use of routine techniques

used in biomineralisation, Coronado et al. (2013) have

demonstrated the presence of primary skeletal structures in

a skeleton of a Syringoporicae from Carboniferous of

Spain. The importance of control about crystallographic

orientations exerted by organisms is crucial to understand

the production and formation of biominerals and the

processes of diagenesis that change the original crystal-

lographic information (Perez-Huerta et al. 2007). In this

regard, several techniques have been systematically used

including polarised microscopy (Schmidt 1924; Burdick

and Mauck 2011), X-ray diffraction (Checa and

Rodrıguez-Navarro 2005; Rodrıguez-Navarro 2006),

transmission electron microscopy (Griesshaber et al.

2009; Lee et al. 2012), electron-backscattering diffraction

(EBSD) (Perez-Huerta et al. 2007; Coronado et al. 2013)

and polarisation-dependent imaging contrast (PIC) map-

ping done using X-ray photoelectron emission spectro-

microscopy with synchrotron radiation (Gilbert et al.

2011; Gilbert 2012).

The scope of this study was the introduction of a new

technique, computer-integrated polarisation (CIP)

microscopy, for the crystallographic analysis of biominer-

alised structures in fossils, and its application in the

detection of diagenetic alteration. CIP has been described

as a method for texture analysis and optical orientation

imaging (Heilbronner and Pauli 1993; Heilbronner 2000;

Heilbronner and Barret 2014). This technique determines

the c-axis orientations of uniaxial minerals from optical

micrographs and displays the results in the form of pole

figures and c-axis orientation images (COIs), using a

colour-code scheme [colour look-up table (CLUT)], which

represents each orientation in two elements (azimuth and

inclination). CIP has beenmainly used in structural geology

for the analysis of quartz and calcite with good results (van

Daalen et al. 1999; Trullenque et al. 2006), but without

application to recent and fossil biominerals.We present this

tool as an alternative to conventional crystallographic

methods used in biomineralisation, showing their advan-

tages and disadvantages in relation to other techniques and

their applications in palaeontology. For this purpose, a

structural and crystallographic case study of skeletons of

Sinopora sp. is presented, including a comparison of

crystallographic data obtained by CIP and EBSD.

Materials

Colonies and colony branches of Sinopora sp. Sokolov,

1955 come from Cantabric Zone (Figure S1(A)), north part

of Iberian Massif (Lotze 1945), and more specifically in

the Vergano Fm., Pisuerga-Carrion Unit, Palencia, Spain

(Figure S1(B)). Samples from Celada de Roblecedo Sur

section were collected (van der Graff 1971), which is

located in the Celada de Roblecedo Anticline between the

Redondo Syncline and the Castillerıa Syncline. It is Late

Moscovian (Myachkovian horizon) in age. Details about

the locality and palaeoecology of taxa are contained in

Coronado and Rodrıguez (2014).

Sinopora Sokolov, 1955 is a tabulate coral that belongs

to the Order Auloporida and Family ?Sinoporidae

Sokolov, 1995. We include provisionally this genus in

the superfamily Syringoporicae because of its similarities

with Multithecopora (Hill 1981). Sinopora shows a small

bushy colonial morphology with cylindrical corallites,

slender, elongated and curved, but with deeper calices with

sharp edges (Figure 1(A)–(C)). Lateral increase, hola-

canth septal spines and tabulae are rare (Figure S1).

Methods

To characterise and evaluate the original preservation of

coral skeleton, a combination of different structural and

crystallographic techniques was used, including petro-

graphic microscopy, scanning electron microscopy (SEM),

atomic force microscopy (AFM) and cold cathodolumi-

nescence (CL). In addition, selected samples were prepared

for their study by EBSD and their observation by

petrographic microscopy and analysed by CIP.

Structural characterisation

SEM and petrographic microscopy were used for

visualisation and characterisation of the microstructures

in the corals. The samples were cut, polished and etched

with formic acid 2% solution for 7 s for SEM. Images were

obtained with a JEOL JSM-820 scanning electron

microscope operated at 20 kV, equipped with an EDX

system, located in the Geological Techniques Research

Assistance Centre (Universidad Complutense de Madrid,

Spain). Thirty-one thin sections of six coral colonies and

two ultra-thin sections were prepared to study by

petrographic microscope and CIP analysis.

The rock counterpart of thin sections that presented

better preservation characteristics were selected to study

the samples with AFM. The different rock pieces were

polished and cut by a circular diamond cutter forming thin

slides of rock. The thin slides were cut and etched with a

Milli-Q water solution for 7 h and observed with an Atomic

Force Microscope [model Digital Instruments Nanoscope

IIIA (Veeco), located at the National Centre of Electron

Microscopy, Universidad Complutense de Madrid, Spain].

The images were obtained at room temperature and in air

using a tapping mode with a silica tip. The images were

processed with the Nanoscope software v5.30 r3 sr3 of

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Veeco Instruments Inc. and the WSxM v5.0 Develop 5.0

software of Nanotec (Horcas et al. 2007). Two main types

of images can be obtained with AFM: height and phase.

Height images enable topographical reconstruction of the

crystal surface, while phase images provide information

regarding the variations inmaterial properties.More details

concerning the properties of each image is presented in

Coronado et al. (2013).

The CL study was carried out with a cold CL probe CL

8200 MK 4 system made by Cambridge Image

Technology Ltd (Cambridge, UK), working at 16 kV and

a current of 0.5mA, fitted to a petrographic microscope

Nikon Eclipse E400 POL with camera, located in the

Geological Storage Division, Hidrogeochemical Group,

Ciemat (Madrid, Spain). CL microscopy has been applied

in the past with the aim to prove the diagenetic or biogenic

origin of coral microstructures (e.g. Mas and Rodrıguez

1990; Fedorowski 2003; Stolarski et al. 2007).

Crystallographic characterisation

For EBSD analyses, samples were prepared using a thin

slide of rock that contains a colony in transversal and

longitudinal sections. The slides were polished with

alumina of 1 and 0.3mm and finally polished with colloidal

silica (0.06mm). Before analysis, sampleswere coatedwith

a thin layer (1.5 nm) of Au/Pt (Perez-Huerta and Cusack

Figure 1. (Colour online) (A–C) Petrographic micrographs of several sections of Sinopora. (A) Transversal section of Sinopora forminga boundstone in bioherm facies. (B) Transversal and longitudinal sections of Sinopora in a boundstone facies truncated by an energeticevent and subsequent recovery of the growth of colony. (C) Cross-polarised image that shows a transversal section of Sinopora. Whitedash lines highlight the fibrous domains and delimit the big middle lamellae domain. Extinction pattern is shown by four complementaryregions, each one of 408 of development. (D–F) SEM images of Sinopora coral wall. (D) Transversal section of Sinopora where thesilicification areas (white arrows), the micritic lumen (L) and the surrounding matrix (M) are observed. (E) Detail of silicification area (Si)in contact with coral wall (Cw). Note the preservation of lamellar and fibrous microstructure. (F) Detail of lamellar sclerenchyma. Notethe micropores (white arrows) scattered in the coral wall. (G, H) CL images. (G) Transversal section of a corallite. Note the brightluminescent of matrix and lumen areas. White arrow points to alteration zones that begin in the luminiscence external fibrous domain andprogress by the lamellar sclerenchyma as a random pattern. (H) Transversal section of a corallite with two lateral buddings. Note the NLareas in the middle of coral wall and the L areas in borings (white arrows) and in the two fibrous domains. (I) Tangential sections of twocorallites. Note the dull and brilliant luminescence of fractures and micro-fractures that cut the coral wall and the great alteration areas intheir edges of corals. These alterations zones present inner micro-cracking.

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2009). The EBSD study was carried out with an Oxford

Nordlys camera mounted on a Field Emission Scanning

Electron Microscope JEOL 7000 located in the Central

Analytical Facility (CAF) of the University of Alabama.

EBSD data were collected with the Oxford Aztec 2.0

software at high vacuum, 30 kV, large probe current and a

resolution of 0.3mm step size for crystallographic maps.

Finally, data were analysed using OIM 5.3 from EDAX-

TSL. In this study, EBSD data are represented by

crystallographic maps and pole figures, which represent

the stereographic projection of crystallographic planes in

reference to the {0001}, f10�10} and f01�10} calcite planesin a transversal section of Sinopora sp.MATLABe toolbox

MTEX (Bachmann et al. 2011) has been used with the

purpose to plot misorientations.

For CIP analysis, two ultra-thin sections, representing

transverse and longitudinal sections, of Sinopora sp. were

used. For the analysis of thin sections, it is necessary to use

ultra-thin sections due to the size of crystals, with better

results obtained when the first grey order of interference

colour is reached during polishing. In some cases, the

ultra-thin sections in the limit with second order of

interference colour (blues and yellows colourations) can

also be used. The method of preparation of ultra-thin

sections was described by Lafuste (1970) and the samples

should be between 2 and 10mm in thickness. This

thickness induces some alterations in the CIP analysis, due

to impurities in the glass (dust, scratches or particles), and

it requires careful cleaning of the surfaces before analysis.

Six CIP analyses in different corallites were performed

with a petrographic Zeiss microscope with a reflex camera

attached and a Kodak Wratten filter no. 25, at 20 £ and

40 £ magnifications, to determine the crystallographic

correlation of coral wall microstructure and were

processed with the Image SXM software (Barret 1997).

The resolution is 3072 £ 2048 pixel and the ratio pixel–

mm is 1:1.14.

Misorientation images were analysed to characterise

the potential diagenetical alteration of these coral samples.

These images show the misorientation, which is the

number of degrees required to bring these two crystals into

crystallographic alignment on a common axis, for each

pixel measurement in north–south and east–west direc-

tions. In addition, circular polarisation images (details in

Heilbronner and Barret 2014) were obtained to distinguish

some regions affected by recrystallisation. Finally, with

the purpose of measuring the misorientations among

crystals and establishing a comparison with EBSD data, it

was necessary to make a transect of misorientations using

the SXM Image, measuring the difference of orientation

between two correlated crystals. This analysis was carried

out using a random process of measure during 200 contacts

of crystals, helped by the azimuthal image of COI and

plotting the results in a histogram.

Results

Structure of Sinopora

Microstructural characterisation

Sinopora skeletons are formed by alternating domains of

calcite crystals, with two morphologies: lamellae and

fibres. The microstructure is composed of three domains,

an external fibrous domain, a middle lamellar domain and

an inner fibrous domain (Figure 1(C)), with fibres located

within the external and inner domains. Occasionally, the

preservation of coral does not clearly permit to observe the

fibrous domains, with some corals showing signs of

abrasion at their external edges, resulting in the removal of

the fibrous domain.

Fibres (Figures 4(A), 6(A) and 8(A)) are elongate

crystals, with length from 12 to 30mm (x ¼ 19mm) and

width between 2 and 5mm (x ¼ 3mm), with irregular

morphology and indentations to their edges. The morpho-

logical axis is permanently oriented towards the lumen,

perpendicular to growth direction. A gradual transit between

fibres and lamellae is produced, as it is characteristic in the

Palaeozoic corals (Semenoff-Tian-Chansky 1974; Lafuste

1978, 1983; Rodrıguez 1989; Falces and Rodrıguez 1999).

Sometimes, it can be recognised in Sinopora skeleton, a

branch-off in the transition point where two or more fibres

form a lamellae (Figure 6(A)) or lamellae which turn 908their morphological axis (L-shaped) and point to the lumen

in the transit to fibrous domain (Figures 6(A) and 8(A)).

Lamellae, sensu Lafuste (1983), are the second

morphological element that make the microstructure in

Sinopora and are the most abundant. Lamellae are straight

to slightly wavy, with indentations at their edges, and are

completely imbricated with each other showing a compact

packaging (Figure 1(F)), being the most characteristic

morphology the ‘scutellate’ lamellae (Lafuste 1983).

These structures having a lateral development in two

dimensions, with lengths from 5 to 30mm (x ¼ 16mm)

and width from 2 to 4mm (x ¼ 3mm), are microlamellae,

sensu Rodrıguez (1989). The morphological axis is

permanently oriented perpendicular to the lumen, parallel

to growth direction. The concave part of lamellae always

points out to the lumen, showing a concentrically aspect of

the lamellae domain (Figure 1(C),(F)). Petrographic

microscopy shows an undulate extinction, around the

coral wall, which is characteristic of Syringoporicae. The

extinction in a transversal section shows a cross with four

extinct regions at 408 (Figure 1(C)).

Nanostructural characterisation

AFM is a powerful technique to observe the original

nanostructures of fossil skeletons and their preservation

state. This method have been applied in tabulate (Coronado

et al. 2013) and scleractinian (Stolarski and Mazur 2005)

corals with remarkable results. AFM images of the coral

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wall (Figure 3(B)–(D)) show an intricate nanostructure

formed by nanogranules in both lamellae and fibres

(Figure 4). The nanogranules are mainly rounded and very

well rounded, with irregular morphologies. The sizes are

more variable, 31–74 nm (x ¼ 46 nm), but the distribution

is random (Figure 4), in contrast to other Syringoporicae

where the nanotexture is co-oriented (Coronado et al.

2013). The edges of the nanogranules exhibit a dark colour

in phase images, indicating that they have a different

composition (Figure 2(D),(E)). In topography image, some

nanogranules present a rim around them, with other

topography indicating that the compositional variation is

part of other phase or a mixing of phases. Several authors

(Dauphin 2002; Cuif et al. 2008; Gorzelak et al. 2013) have

studied these envelopes in recent and fossil organisms and

proposed that they could be organic coatings from the

original organic matrix.

Coral wall preservation

There are some signs of diagenetical alteration observable

by CL, SEM and AFM. Skeletons of Sinopora show some

good preservation of their original morphology and

structure, but some corallites show patterns of alteration.

Corals by CL exhibit in the majority of them null or low

luminescence, showing a distribution of the luminescence

mainly in the fibrous domains of the external and inner

edges. The luminescence of the matrix is very bright

(Figure 1(G)). Bright luminescence is common in fractures

of the rock (Figure 1(I)) and micro-cracking of the

skeletons in borings (Figure 1(G),(H)). Dull luminescence

is common in coral wall from the external part as a

migration of fluids, and later, recrystallisation in these

areas is observable in a scattered pattern of luminescence.

On the other hand, there are some corals that present bright

luminescence in regions of the coral wall, associated to

micro-cracking (Figure 1(I)) and later alteration from

these points.

SEM also show alteration signs in the coral walls,

with a minimum number of corals altered by silicification

on their edges. This pattern of alteration is produced by

the migration of fluids from the external part of coral to

the inner one, but without reaching the lumen. Sometimes,

it is observable in little gulf around the silicification areas,

the microstructure preserved or partially preserved around

of them (Figure 1(E)). In these last areas, a degrading

neomorphism pattern is observable around the original

microstructure. Likewise, it is common that some corals

presenting an alteration pattern by dissolution in the form

Figure 2. (Colour online) (A–F) AFM images of a coral wall of Sinopora. (A) Height image of the longitudinal section of a lamella. (B)Phase image of A where granular-shaped nanocrystals are observed. Note the thickness of the dark coating around crystals. (C) Heightimage of the longitudinal section of a lamella. (D) Phase image of C where granular-shaped nanocrystals and the partial merging betweennanocrystals by recrystallisation are observed. (E) Height image of the longitudinal section of a lamella. (F) Phase image of E. Note theflattening of the topography in the height image and the loss of nanotexture in the phase image by recrystallisation.

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of isolated pores scattered by the wall (Figure 1(F),

white arrow).

AFM data exhibit some regions in the external part

of coral in which nanogranules are melted, losing the

original morphology and increasing their size (Figure 4

(B),(C),(E),(F)). In these areas, the dark-coloured coating

observable in the coral wall is lost or partially lost by

this nano-recrystallisation, in correspondence with the

luminescence by CL.

Crystallography of Sinopora

Electron-backscattering diffraction

EBSD data confirm observations of microstructure by

petrographic microscopy and SEM, in addition, to provide

crystallographic information (Figure 3). Diffraction maps

of transversal section in the middle zone show well-

defined crystals with microlamellae morphology (Figure 3

(B)), as well as reflects that ‘scutellate’ microlamellae can

be imbricated composing a frame (Figure 3(B)).

Crystallographic maps indicate that each structure

(lamellae in this case) behaves like a single crystal (Figure 3

(C)). The lamellae present a c-axis of calcite perpendicular

to their elongation, with an inclination respect to the

morphological axis of ca. 308. It is characteristic that the

distribution of colours derived by preferred crystal-

lographic orientations show packages of lamellae with the

same orientation, cut by other packages with a different

orientation. Pole figures of three plane of calcite f10�10},f01�10} and {0001} are represented, with the purpose of

obtaining the distribution of orientation of lamellae in each

plane. The plane {0001} shows a unique pole maxima with

all the orientations located in the same direction of c-axis,

with an inner variation of 608. In contrast, the f10�10} and

f01�10} planes show a gradual rotation of a and b axes

around c-axis, with the last one fixed.

The misorientation has been analysed with the MTEX

tool, which measures the different crystallographic

orientation between two grains (crystals) which are in

contact. A grain map, of EBSD crystallographic map, was

done using a threshold of 28, in order to reduce the noise inthe inner variation of each crystal. Subsequently, a

misorientation histogram was generated to indicate that

the main order of misorientation is less than 58, with a

mode of ca. 2.58 and the second most common is ca. 158.The next maxima of misorientation are ca. 358, 408 and 558(Figure 3(D)).

Computer-integrated-polarisation

Six COIs and their corresponding pole figures were

obtained using the CIP method from different corallites of

two ultra-thin sections. Figure 4 represents a view of a

transverse section of Sinopora from the middle lamellar

domain to the inner fibrous domain. The petrographic

micrograph (Figure 4(A)) shows a main lamellar

sclerenchyma in transit to a fibrous domain in a second,

blue order of colour of interference. The orientation image

(Figure 4(B)) shows four main colourations. Pale blue

tones are characteristic of the lamellar tissue where the

c-axis is oriented perpendicular to morphological axis and

parallel for the fibres, which indicate a gradual transition,

sharing the orientation of c-axis similar to that observed by

EBSD in Multithecopora hontoriense (Coronado et al.

2013). The pole figure (Figure 4(E)) shows a unique

maxima pole, with a wide distribution of 608, similar to

data obtained by EBSD and a c-axis slightly tilted ca. 35–

408 versus the morphological axis. Red and yellow colours

show other orientations that are present in the coral wall

only in altered areas. Some fibres in region A2 maintain

the microstructure, whereas in region A1 (Figure 4(B))

there is loss of microstructure probably due to a degrading

neomorphism. Both areas reflect random orientations in

the pole figures (Figure 4(E)). The misorientation image

shows green and blue colourations to those crystals with

the same orientation, whereas the deep purple and yellow

colourations sign those crystals that have lost the original

orientation and present a random distribution in pole figure

probably due to diagenesis, as is shown in Perez-Huerta

et al. (2007) and Moreno-Azanza et al. (2013).

A detail of microstructure in a transversal section is

shown in Figure 5. Petrographic micrograph shows a

lamellar region with scutellate lamellae, which have

irregular morphologies and wavy edges (Figure 5(A)).

COI (Figure 5(B)) shows a main orientation perpendicular

to morphological axis with main pole maxima to the lumen

and an internal variation of ca. 608. The variation of

orientation, shown by the colours of COI, reflects a similar

distribution that is obtained by EBSD result, where the

crystals with same orientations are interconnected forming

a unique patch (green and yellow bundles). Larger black

spots are impurities of the thin sections (such as bubbles

and dust) that potentially produce errors in the

interpretation of the image, but they have been removed

by the software (Heilbronner and Barret 2014).

A histogram of misorientation distributions has been

constructed by a manual measurement of misorientations

at the contact of crystals. The histogram, of 58 binning,shows a normal distribution of misorientation angle of

lamellae for this region. The main misorientation is 108followed by 158, 208, 358 and 558. Similar results have

been described by EBSD. The unique differentiation is

that there is a maximum between 08 and 58, which can be

explained by the formation of patches of crystals co-

oriented. CIP method has a crystallography sensitivity

greater than 108 (Trimby and Prior 1999), although we

have detected some little variations above 58, the

differentiation between crystals with the same orientation

or a variation less than 58 is very difficult with this

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methodology. The azimuthal image, derived from COI

data, permits to distinguish better the morphology of

crystals and reminds that of the diffraction map of EBSD.

This image helps us to distinguish, in combination with

petrographic micrographs, those crystals with a misor-

ientation greater than 58.

Figure 6 represents a view of a tangential section,

almost longitudinal. A great development of inner fibrous

domain versus the external can be observed in

petrographic micrograph (Figure 6(A)). The fibrous

external domain is surrounded by micritic matrix (M).

Lamellar domain show two main directions derived from

Figure 3. (Colour online) (A) Image that shows longitudinal and transversal sections of skeleton of Sinopora sp. and the location of theEBSD maps (white rectangle). (B, C) Crystallography by EBSD of coral wall in a transverse section of corallite. (B) Index intensity map,showing the structure lamellar tissue of coral wall. (C) Crystallographic orientation map, showing the main crystallographic orientationsof coral wall. (D) Pole figures [in normal direction (ND) view to the sample surface in a three-axes reference system with the indication ofthe reference (RD) and transverse (TD) directions] indicating crystallographic orientations of calcite crystals with reference to the c-axes,and crystallographic key indicating colour coding of crystallographic axes: a, orientations of lamellae of coral wall in B and C in the plane{0001}; b, orientation distribution in the plane f01�10}; c, orientation distribution in the plane f10�10}. Bar diagrams show the relativefrequency (%) of the misorientation angles.

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tangential cut and the gradual transit to inner fibrous

domain. COI (Figure 6(B)) shows several main colour-

ations (green-red and green-purple). The pole figure shows

two pole maxima in a concordant orientation, turning 408,in a gradual change (Figure 6(C)). Some crystals, in the

contact region of two crystallographic domains have a

transitory orientation between both domains. These two

domains remind the variations observed in the polarised

image (Figure 1(C)), in which there is a polarised area at

408. Matrix crystals have a random orientation and those

fibrous crystals near these areas have lost the original

orientation by diagenetic alteration, showing a random

orientation in the pole figure (Figure 6(C)). It is

remarkable that the loss of thickness, during polishing,

in the ultra-thin section produces a variation in c-axis

interpretation by the software. As shown in the left part of

image (Figure 6(B)), the colourations are more light,

whereas in the pole figure (Figure 6(C)) the main pole

direction is preserved but the inclination has changed,

being this most vertical.

A transversal section of a skeleton of Sinopora is

showed in Figure 7. This was chosen due to the partial

silicification of the external fibrous domain (Figure 7(A)).

In this example, the COI shows a common distribution in

transversal sections (L1), with pole maxima towards the

lumen, the gradual transit between fibres to lamellae and

the rotation of the sclerenchyma tissue of 408 (L2). Thesilicification area (A) shows a random distribution of its

orientations. The lamellae and fibres close to this area kept

the orientations and morphologies despite of the evident

alteration, corroborating the SEM data (Figure 1(E)).

Figure 8 was chosen to show the behaviour of the

inorganic sparitic crystals of calcite precipitated in the

lumen of Sinopora and their products of alteration.

Petrographic micrograph (Figure 8(A)) shows a tangential

section of Sinopora in the contact of lamellar domain with

Figure 4. (Colour online) CIP images and derived c-axis pole figures of a transversal section of Sinopora sp. (A) Ultra-thin sectionimage of the investigated area. (B) Orientation image. Black regions correspond to the thick regions of contact between crystals andopaque impurities, and they were not considered for the c-axis pole figure calculation. A1, alteration zone 1; A2, alteration zone 2; L,lamellar zone. (C) Circular polarisation image. (D) Misorientation image in false colour, showing the variations of colouration due todiagenetical alteration. There are different patterns of colourations from the fibrous inner domain to the external domain (yellowcolourations). (E) Pole figure of the entire area calculated as an orientation distribution function provided in multiples of uniformdistribution intervals of 0.5 for c-axis orientations. Pole figures of different areas of detailed study superimposed on the whole pole figure.Red points correspond to the punctual c-axis orientation of each pixel. L, A1 and A2 correspond to the areas mentioned above. Note therandom distribution in the altered areas versus the lamellae zone. Standard CLUT is in the lower region.

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Figure 6. (Colour online) CIP images and derived c-axis pole figures of a tangential section, almost longitudinal of Sinopora sp. (A)Ultra-thin section image of the investigated area. (B) Orientation image. Black regions correspond to the thick regions of contact betweencrystals and opaque impurities, and they were not considered for the c-axis pole figure calculation. L1 and L2, lamellar zones; M, matrixzone. (C) Pole figure of the entire area calculated as an orientation distribution function provided in multiples of uniform distributionintervals of 0.5 for c-axis orientations. Note the rotation of 408 of cluster of lamellae by the natural rotation of skeleton. (L1–M) Polefigures of different areas of detailed study superimposed on the whole pole figure. Red points correspond to the punctual c-axis orientationof each pixel. L1, L2 and M correspond to the areas mentioned above. Note the random distribution in the matrix area versus the lamellaezones. Standard CLUT is in the middle of image.

Figure 5. (Colour online) CIP images and derived c-axis pole figures of a transversal section of Sinopora sp. (A) Ultra-thin sectionimage of the investigated area. (B) Orientation image. Black regions correspond to the thick regions of contact between crystals andopaque impurities, and they were not considered for the c-axis pole-figure calculation. Note the match with the contact between crystalsand their similar distribution to the non-diffracted areas of EBSD. (C) Pole figure of the entire area calculated as an orientation distributionfunction provided in multiples of uniform distribution intervals of 0.5 for c-axis orientations. Standard CLUT is in the lower region.Histogram of correlated misorientation calculated by punctual measurement between two crystals. (D) Azimuthal image derived fromgeneration process of COI.

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the inner fibrous domain and their relation with the cement

of lumen. The cement of lumen is formed by blocky

cement (C) which has a random orientation (Figure 8(E)).

On the other hand, the skeleton shows a classical

distribution of a tangential section where the lamellae

and fibres have a common orientation with two pole

maxima towards the lumen, perpendicular to morphologi-

cal axis, in the case of lamellae and parallel in fibres. The

misorientation image (Figure 8(D)) reveals an alteration

pattern from the lumen to the lamellar sclerenchyma. Red

and turquoise colourations point to those crystals that have

been altered by fluid from the lumen. It is clearly a visible

alteration pattern similar to that observable by CL in

Figure 1(G),(H), where in a selective way some crystals

are recrystallised. It is worth highlighting that have been

produced the dissolution and after precipitation during the

cementation of crystals in the external fibrous domain, and

it is characteristic in the COI (Figure 8(B)) but with a

different colouration also observable in the circular

polarisation image (Figure 8(C)). This image reveals that

all the crystals of coral wall have the same phase in

juxtaposition to the cement of lumen.

The last example chosen have an alteration area in the

longitudinal section of Sinopora, as can be observed in

petrographic micrograph (Figure 9(A)). COI (Figure 9(B))

shows a coral wall with a main orientation towards the

lumen and the natural rotation of the crystals of 408. The

lumen has a blocky cement of sparitic calcitewith a random

orientation of their crystals. This coral wall has evident

recrystallised areas (A1 and A2) where there are patches of

alteration and where the random orientations are obser-

vables (Figure 9(E)). Misorientation image shows blue

regions that contrast with the orange-yellow well-ordered

sclerenchyma, these areas are the alteration patches which

move a front of recrystallisation from the lumen to middle

areas. In the left-lower part of COI can be observed a

degrading neomorphism with a random orientation

followed by a big alteration area with red colourations

due to the loss of orientation.

Discussion

Evaluation of CIP as a technique for fossils

The CIP method is a good alternative tool to classical

methods used in the analysis of crystallographic infor-

mation of fossils. The method is able of mapping c-axis

orientation distributions, generate misorientation images

and measure misorientations. In combination with petro-

graphic microscopy, this method is also capable of

distinguishing mineral phases and some properties of

biominerals. In comparison with other techniques, CIP has

Figure 7. (Colour online) CIP images and derived c-axis pole figures of a transversal section of Sinopora sp. (A) Ultra-thin sectionimage of the investigated area. (B) Orientation image. Black regions correspond to the thick regions of contact between crystals andopaque impurities, and they were not considered for the c-axis pole-figure calculation. L1 and L2, lamellar zones; A, alteration zonearound silicified area. (C) Pole figure of the entire area calculated as an orientation distribution function provided in multiples of uniformdistribution intervals of 0.5 for c-axis orientations. (L1–M) Pole figures of different areas of detailed study superimposed on the wholepole figure. Red points correspond to the punctual c-axis orientation of each pixel. L1, L2 and A correspond to the areas mentioned above.Note the random distribution in the altered area versus the lamellae zones and the natural turn of 408 of lamellae cluster. Standard CLUT isin the middle of image.

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a disadvantage related to the fact that it can only be used in

uniaxial minerals (e.g. calcite, quartz and hydroxylapatite),

but not in biaxial minerals, such as aragonite. Although the

method can be used for many recent and fossil uniaxial

biominerals, which compose monomineralic skeletons

such as foraminifera, radiolarian, cnidarian, porifera,

molluscs, brachiopods, bryozoans, algae, egg-shells and

chordate skeletons, among others (Lowenstam and Weiner

1989), it limit its uses in bimineralic and polymineralic

biomineral structures in some organisms including

molluscs (Dalbeck et al. 2006; Perez-Huerta et al. 2011),

some bryozoans (Taylor et al. 2008; Benedix et al. 2014),

annelids (Ries 2011), crustaceans (Al-Sawalmih et al.

2008), as well as those formed basically by biaxial minerals

as scleractinian corals (Dauphin et al. 2006) and

cephalopods (Dauphin 2002). This limitation of CIP to

uniaxial minerals is a major drawback in contrast to the

application of EBSD.

The CIP method has a good spatial resolution, which

depends on the resolution of camera (in our study, 90 nm at

40 £ of magnification) but lower than that of EBSD

(20 nm), and PIC mapping, which offer 10 nm of

resolution, although it provides in situ data that are not

the case for X-ray diffraction. The limitation of c-axis by

CIP is evident against the versatility of textural methods

including EBSD or X-ray diffraction textures, which

permit the analysis of multiple crystallographic planes,

although other techniques also measure only the c-axis

orientation as PIC mapping (Gilbert et al. 2011; Gilbert

2012) and they are less accessible for routine analysis.

The main strength of the CIP method is that it can be

used as a complementary tool when working with known

organisms in which some crystallographic information is

already available. CIP use can help to distinguish growth

mechanisms of biominerals in fossils and to assess their

state of preservation. A major advantage of CIP is the rapid

Figure 8. (Colour online) CIP images and derived c-axis pole figures of a tangential section of Sinopora sp. (A) Ultra-thin section imageof the investigated area. (B) Orientation image. Black regions correspond to the thick regions of contact between crystals and opaqueimpurities, and they were not considered for the c-axis pole-figure calculation. L, lamellar zone; A, alteration zone; M, matrix zone. (C)Circular polarisation image of studied area. Note the common phase detected by this image in ultra-thin section in the first order in thecoral wall versus the great variation of the matrix. (D) Misorientation image in false colour. Note the random colourations from the matrixversus the green colouration of unaltered areas. The altered areas by diagenesis show red and yellow colourations. (E) Pole figure of theentire area calculated as an orientation distribution function provided in multiples of uniform distribution intervals of 0.5 for c-axisorientations. (L–M) Pole figures of different areas of detailed study superimposed on the whole pole figure. Red points correspond to thepunctual c-axis orientation of each pixel. L, A and M correspond to the areas mentioned above. Note the random distribution in the alteredand matrix areas versus the lamellae zones. Standard CLUT.

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data acquisition at low and high magnifications, resulting

in fast acquisition of crystallographic data for large areas

(7 £ 5, 3.5 £ 2.4, 1 £ 0.7, 0.5 £ 0.35 and 0.3 £ 0.19mm,

depending of the used magnification). Furthermore, this

methodology does not require special treatment of samples

when ultra-thin sections or thin sections are available. In the

case of biominerals of hundreds of micrometres of size, i.e.

egg shells (Moreno-Azanza et al. 2013) or belemnites

(Dauphin et al. 2007), thismethodology can be appliedwith

thin sections. Finally, many museum samples of fossils, for

which destructive techniques are not allowed, are

represented by thin sections of materials, representing

ideal target samples for the use of the CIP methodology.

The data of Sinopora presented in this study show that the

CIPmethod have a good correlationwith the data offered by

EBSD and earlier results presented for another fossil coral,

Multithecopora hontoriense (Coronado et al. 2013).

Similar data have been shown in comparison with EBSD

and CIP in structural geology (van Daalen et al. 1999;

Heilbronner and Barret 2014).

Characterisation of preservation state of fossil samples

Many palaeoclimatological and palaeoenvironmental

studies are based on the use of biogenic skeletons to

obtain the short- and long-term environmental variations as

palaeotemperature or palaeosalinity (Grossman et al. 1996;

Wenzel et al. 2000; Mii et al. 2001; Rosales et al. 2004).

These studies are based on the premise of the skeletons

record the geochemical signature in which they were

precipitated and are founded from high-resolution isotope

and trace element examination of growth profiles of marine

organism (both invertebrates as vertebrates). To reach this

purpose, the geochemical signatures depend on the

ultrastructural integrity and crystallinity after diagenesis

(Perez-Huerta et al. 2012). Even so, some palaeontological

studies are also supported by the preservation state of

fossils (Simoes et al. 2009; Dzik et al. 2010; Grellet-Tinner

et al. 2011; Coronado et al. 2013); consequently, the

microstructural characterisation can be of phylogenetic

importance (Burrow and Turner 2012; Mikhailov 2013;

Moreno-Azanza et al. 2014). Several techniques have been

Figure 9. (Colour online) CIP images and derived c-axis pole figures of a longitudinal section of Sinopora sp. (A) Ultra-thin sectionimage of the investigated area. (B) Orientation image. Black regions correspond to the thick regions of contact between crystals andopaque impurities, and they were not considered for the c-axis pole-figure calculation. L, lamellar zone; A1, A2, alteration zones. (C)Circular polarisation image of studied area. Note the common phase detected by this image in ultra-thin section in the first order in thecoral wall versus the variation in the altered areas. (D) Misorientation image in false colour. Note the blue colourations from the alterationareas versus the red–yellow colouration of unaltered areas. (E) Pole figure of the entire area calculated as an orientation distributionfunction provided in multiples of uniform distribution intervals of 0.5 for c-axis orientations. (L–A2) Pole figures of different areas ofdetailed study superimposed on the whole pole figure. Red points correspond to the punctual c-axis orientation of each pixel. L, A1 andA2 correspond to the areas mentioned above. Note the random distribution in the altered versus the lamellae zones. Standard CLUT.

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used to help characterise the preservation state of fossils

skeleton, such as scanning microscopy, CL, X-ray

diffraction, transmission electron microscopy coupled

and ultimately geochemical analysis, which help to

discriminate those analyses that are ‘outlaws’ in the

variation searched (Armendariz et al. 2008; Lee et al.

2012). Presumably with this focus on mind, some research

groups began to improve the techniques used to

characterise the state of preservation of fossil, implement-

ing techniques such as EBSD, which offer a new

perspective in this field, characterising the crystallography

of skeletons and offering a new perspective of diagenetical

alteration (Perez-Huerta et al. 2007; Stolarski et al. 2007;

Coronado et al. 2013).

One of most used techniques in the characterisation of

preservation state is CL, which is particularly valuable in

the sedimentary petrology area (Richter et al. 2003; Boggs

and Krinsley 2006). The behaviour of marine carbonates

under CL is usually a good indicator of diagenetic alteration

by burial, and it is applied to identify recrystallisation

processes. In calcium carbonate material, the variation of

CL is generally attributed to the concentration ratio of

the activator, which is incorporated in the skeletons

during digenesis (Rosales et al. 2004). Pb2þ, Mn2þ and

trivalent rare-earth elements ions are regarded as the main

activators to luminescence in calcites, whereas Fe2þ is the

main inhibitor (Richter et al. 2003; Flugel 2004; Barbin

2013). As a result, non-luminescent (NL) calcites are

considered unaffected by diagenesis. A geochemical

analysis of these elements in the samples can help to test

the existence of diagenetic alteration. However, the

previous premise is questionable in biogenic carbonates

due to the incorporation of little amounts of Mn2þ or Fe2þ

by metabolic activity or/and by physicochemical pattern of

the surrounding environment during their growth and

ontogeny (Barbin et al. 1991; Barbin and Gaspard 1995;

Barbin 2000; Barbin 2013), and also some of biogenic

carbonates can be diagenetically affected and NL, because

they have no scavenged activators in the new crystals,

considering that, for example, some silicification areas are

NL and their surrounding alteration is alsoNL (unpublished

results). With these reasons derived, CL is usually

complemented with geochemical data (Grossman et al.

1996; Rosales et al. 2004). As shown by the results

presented in this study, the crystallographic methods can

complement this characterisation, and the CIP method

permits to have a combination of both techniques

(Heilbronner and Barret 2014), which offer a double vision

giving crystallographic information together with geo-

chemical by CL. The combination of both or more

techniques permits to get a better characterisation of

preservation state of biogenic skeletons.

Within the current study, SEM, CL and AFM images

reveal the presence of some altered areas by diagenesis.

The CL images of whole corals show a visible preference

of alteration of fibrous domains and random pattern of

alteration on lamellar domain. These patterns of alteration

are similar to that observed by COI, where the alteration

front advances form the external and internal areas,

modifying isolated crystals or cluster of crystals of tens of

microns. These patterns have a very good correlation with

the AFM data, where some isolated regions of hundreds of

nanometres show signs of alteration (merging nanogra-

nules and loss of dark coatings), but this alteration is

non-continuous on the contrary as was described in

Multithecopora hontoriense (Coronado et al. 2013). SEM

images expose some alteration arrays as are dissolution

pores, and silicification. The surrounding region micro-

crystals of silicified areas occasionally show degrading

neomorphism and in some cases kept the biogenic

morphology. The COI images show that these last areas,

where the microstructure is also kept, saved the crystal-

lographic orientation in most regions.

Conclusions

CIP method has been applied satisfactorily in the study of

fossils skeletons of Sinopora. The study includes a

previous characterisation of sample by SEM, AFM and

CL with the purpose of distinguishing the diagenetical

alteration. Later, a crystallographic comparison between

CIP and EBSD has been made getting a very good

correlation between the two methods. The CIP method

allows obtaining COIs, pole figures, and measure and

mapping the misorientation. CIP have some advantages

and disadvantages that limit and promote their use, with

respect to the other crystallographic techniques applied in

palaeontological studies.

. This technique can only be used in uniaxial

biominerals (calcite, quartz and hydroxylapatite),

limiting its use for biaxial or bimineralic and

polimineralic biominerals.. CIP method has good spatial resolution (limited by

camera); in our example, 90 nm versus 20 nm of

EBSD.. CIP method can only analyse a unique crystallo-

graphic plane, which is the one containing the c-axis.. It does fast acquisition of data in low and high

magnifications.. This method does not require special treatment of

samples and can be very useful for the analysis of thin

and ultra-thin sections.. It is a technique very affordable for any research

centre.. It is very useful in museum samples where only non-

destructive techniques are allowed.. CIP method detects very fast diagenetic alterations,

which combined with CL offers geochemical and

crystallographic information.

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Acknowledgements

This article is a contribution to the Spanish Working Group IGCP596 Climate change and biodiversity patterns in the Mid-Palaeozoic (UNESCO). The authors thank the staff of ResearchAssistance Centre of Geological Techniques and National Centreof Electron Microscopy (Universidad Complutense of Madrid,Spain) and Marıa Jesus Turrero for allowing them to use thecathodoluminescence (CIEMAT). A.P-H. thanks the College ofArts & Sciences (The University of Alabama) for support andJohnny Goodwing (CAF) for help with SEM. The authors thankthe anonymous reviewer and the editor Gareth Dyke for theirhelp and constructive comments on the text.

Funding

This work was supported by the Spanish Ministerio de Economıay Competitividad [research project CGL2012-30922BTE];Complutense University Research Group [910231]; FPI-UCMgrant [Ismael Coronado].

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